WO2019217253A1 - Polypeptides chimériques, molécules d'acide nucléique, cellules et procédés associés - Google Patents

Polypeptides chimériques, molécules d'acide nucléique, cellules et procédés associés Download PDF

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WO2019217253A1
WO2019217253A1 PCT/US2019/030803 US2019030803W WO2019217253A1 WO 2019217253 A1 WO2019217253 A1 WO 2019217253A1 US 2019030803 W US2019030803 W US 2019030803W WO 2019217253 A1 WO2019217253 A1 WO 2019217253A1
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cell
seq
nucleic acid
chimeric
polypeptide
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Stephen WAGGONER
Hermine BRUNNER
Seth REIGHARD
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Cincinnati Childrens Hospital Medical Center
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Cincinnati Childrens Hospital Medical Center
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Priority to EP19729591.8A priority Critical patent/EP3790896A1/fr
Priority to US17/052,894 priority patent/US20240216509A1/en
Publication of WO2019217253A1 publication Critical patent/WO2019217253A1/fr
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/10—Cellular immunotherapy characterised by the cell type used
    • A61K40/11—T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/10—Cellular immunotherapy characterised by the cell type used
    • A61K40/15—Natural-killer [NK] cells; Natural-killer T [NKT] cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/20—Cellular immunotherapy characterised by the effect or the function of the cells
    • A61K40/22—Immunosuppressive or immunotolerising
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/30—Cellular immunotherapy characterised by the recombinant expression of specific molecules in the cells of the immune system
    • A61K40/31—Chimeric antigen receptors [CAR]
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/416—Antigens related to auto-immune diseases; Preparations to induce self-tolerance
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4202—Receptors, cell surface antigens or cell surface determinants
    • A61K40/421—Immunoglobulin superfamily
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    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
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    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70514—CD4
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70517—CD8
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70521—CD28, CD152
    • C—CHEMISTRY; METALLURGY
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    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70532—B7 molecules, e.g. CD80, CD86
    • C—CHEMISTRY; METALLURGY
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    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70575—NGF/TNF-superfamily, e.g. CD70, CD95L, CD153, CD154
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70578—NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70596—Molecules with a "CD"-designation not provided for elsewhere
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0636—T lymphocytes
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0646—Natural killers cells [NK], NKT cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
    • A61K2239/31—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterized by the route of administration
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
    • A61K2239/38—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterised by the dose, timing or administration schedule
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
    • C—CHEMISTRY; METALLURGY
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    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/33—Fusion polypeptide fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies
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    • C12N2510/00—Genetically modified cells
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    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
    • C12N2740/15041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/15043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • follicular helper T cells can be helpful for vaccine and infection elicitation of long- lived humoral immunity, but exaggerated TFH responses can sometimes promote certain diseases, such as autoimmune diseases (e.g., Sjogren’s syndrome, juvenile
  • TFH cells also appear to play roles in allergies (e.g., IgE related allergies) and cancer (e.g., lymphoma).
  • some embodiments of the invention address one or more of the deficiencies described above.
  • some embodiments of the invention include chimeric polypeptides.
  • Other embodiments of the invention include chimeric nucleic acid molecules encoding a chimeric polypeptide.
  • Other embodiments of the invention include vectors comprising a chimeric nucleic acid molecule.
  • Still other embodiments of the invention include cells comprising a chimeric nucleic acid molecule, a chimeric polypeptide, or both.
  • Yet other embodiments of the invention include methods of making cells. Some embodiments include methods of treating disease. Additional embodiments of the invention are also discussed herein.
  • Some embodiments of the invention include a chimeric polypeptide wherein the chimeric polypeptide comprises (a) an extracellular segment comprising PD- Ll (Programmed Death-Ligand 1) or a portion of PD-L1, or one or more modifications thereof, (b) a transmembrane segment, and (c) an intracellular segment comprising a first signaling polypeptide and optionally one or more second signaling polypeptides.
  • the PD-L1 is a mammalian PD-L1, a mouse PD-L1, a dog PD-L1, a cat
  • the extracellular segment comprises SEQ ID NO: 14 or SEQ ID NO: 15, or one or more modifications thereof.
  • the extracellular segment further comprises one or more spacer sequences, where each of the one or more spacer sequences comprises from 5 amino acids to about 1000 amino acids.
  • the extracellular segment further comprises one or more spacer sequences and the one or more of the spacer sequences comprises CD8a, a portion of CD8a, a hinge CD8a sequence, a leader CD8a sequence, CD8P, a portion of CD8P, CD4, a portion of CD4, CD28, or a portion of CD28, or one or more modifications thereof, or combinations thereof.
  • the extracellular segment comprises one or more of SEQ ID NO: l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5,
  • the transmembrane segment comprises SEQ ID NO: 16, SEQ ID NO: 17, or portions thereof, or one or more modifications thereof.
  • the first signaling polypeptide is O ⁇ 3z, FcRy, FcRp, CD3y, CD35, CD3e, CD5, CD22, CD79a, CD79b, or CD66d, or portions thereof, or one or more modifications thereof.
  • the first signaling polypeptide comprises SEQ ID NO: 18, SEQ ID NO: 19, or SEQ ID NO:20, or one or more modifications thereof.
  • the intracellular segment comprises a second signaling polypeptide and the second signaling polypeptide is CD2, CD4, CD5, CD8a, CD8p, CD28, CD134, CD137, ICOS, or CD154, or portions thereof, or one or more modifications thereof.
  • the intracellular segment comprises a second signaling polypeptide and the second signaling polypeptide comprises SEQ ID NO:2l or SEQ ID NO:22, or one or more modifications thereof.
  • the intracellular segment comprises SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:2l, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, or one or more modifications thereof.
  • the chimeric polypeptide comprises one or more of SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:2l, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:
  • the chimeric polypeptide comprises SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:3l, or one or more modifications thereof.
  • Some embodiments of the invention include a chimeric nucleic acid molecule encoding the chimeric polypeptide as disclosed herein.
  • the chimeric nucleic acid molecule has at least an 80% identity to SEQ ID NO:32.
  • the chimeric nucleic acid molecule has at least an 90% identity to SEQ ID NO: 32.
  • the chimeric nucleic acid molecule encoding the polypeptide is SEQ ID NO: 32.
  • the chimeric nucleic acid molecule is in a cell, an insect cell, a mammalian cell, a human cell, an sf9 insect cell, a rat cell, a mouse cell, a CHO cell, an immune cell, an immune cell progenitor, a T cell, a T cell progenitor, an NK cell, KHYG1, NK-92, YT, SNK-6, NKL or an NK cell progenitor.
  • the chimeric nucleic acid molecule is in an immune cell, an immune cell progenitor, a T cell, a T cell progenitor, an NK cell, KHYG1, NK- 92, YT, SNK-6, NKL, or an NK cell progenitor.
  • the chimeric nucleic acid molecule is in a cell isolated from an animal, a mammal, a primate, or a human.
  • the chimeric nucleic acid molecule is included in a vector, a viral vector, or a plasmid.
  • Some embodiments of the invention include a vector comprising any of the chimeric nucleic acid molecules disclosed herein.
  • the cell comprises any chimeric polypeptide disclosed herein, any chimeric nucleic acid molecule disclosed herein, or any vector disclosed herein.
  • the cell is an immune cell, a stem cell, a mammalian cell, a primate cell, or a human cell.
  • the cell is autologous or allogeneic.
  • the cell is a T cell, a CD8- positive T cell, a CD4-positive T cell, a regulatory T cell, a cytotoxic T cell, or a tumor infiltrating lymphocyte.
  • the cell is an NK cell, an KHYG1 cell, an NK-92 cell, a YT cell, an SNK-6 cell, or an NKL cell.
  • Some embodiments of the invention include a composition comprising any chimeric polypeptide disclosed herein, any chimeric nucleic acid molecule disclosed herein, any vector disclosed herein, or any cell disclosed herein.
  • the amount of the chimeric polypeptide, the chimeric nucleic acid molecule, the chimeric vector, or the cell is from about 0.0001% (by weight total composition) to about 99%.
  • Some embodiments of the invention include a pharmaceutical composition comprising any chimeric polypeptide disclosed herein, any chimeric nucleic acid molecule disclosed herein, any vector disclosed herein, or any cell disclosed herein.
  • the amount of the chimeric polypeptide, the chimeric nucleic acid molecule, the chimeric vector, or the cell is from about 0.0001% (by weight total composition) to about 50%.
  • Some embodiments of the invention include a method of producing any cell disclosed herein comprising adding to a cell any chimeric nucleic acid molecule disclosed herein or any vector disclosed herein. Some embodiments of the invention include a method of producing any cell disclosed herein comprising (a) isolating a first cell, (b) adding to the isolated first cell, any chimeric nucleic acid molecule disclosed herein or any vector disclosed herein, to result in any cell disclosed herein, and (c) optionally recovering and/or expanding the resulting cell from step (b).
  • the first cell is an immune cell, a stem cell, a mammalian cell, a primate cell, or a human cell.
  • the first cell is autologous or allogeneic.
  • the first cell is a T cell, a CD8-positive T cell, a CD4-positive T cell, a regulatory T cell, a cytotoxic T cell, or a tumor infiltrating lymphocyte.
  • the first cell is an NK cell, an KHYG1 cell, an NK-92 cell, a YT cell, an SNK-6 cell, or an NKL cell.
  • Some embodiments of the invention include a method for treating an animal with a disease comprising administering any cell as disclosed herein, any cell produced by as disclosed herein, any cell composition as disclosed herein, or any cell pharmaceutical composition as disclosed herein.
  • the disease is autoimmune disease, allergies, asthma, Sjogren’s syndrome, juvenile dermatomyositis, multiple sclerosis, type-l diabetes mellitus, rheumatoid arthritis, systemic lupus erythematosus (SLE), cancer, blood cancer, solid tumor, leukemia, lymphoma, myeloma, breast cancer, ovarian cancer, glioblastoma, osteosarcoma, medulloblastoma,
  • the disease is autoimmune disease, Sjogren’s syndrome, juvenile dermatomyositis, multiple sclerosis, type-l diabetes mellitus, rheumatoid arthritis, systemic lupus erythematosus (SLE), or cancer.
  • the animal is a mammal, primate, human, mouse, or rat.
  • the administration comprises parenteral administration, a mucosal administration, intravenous administration, subcutaneous administration, topical administration, intradermal administration, oral administration, sublingual administration, intranasal administration, or intramuscular administration.
  • the animal is in need of treatment. [0012] Other embodiments of the invention are also discussed herein.
  • FIG. 1 PD-1 is a selective marker of human TFH cells (A-C) &
  • D-H Development of an embodiment of PD-L1 CAR NK cells (D-H).
  • A Representative PD-l expression levels determined by flow cytometry on electronically gated subsets of leukocytes in non-inflamed human tonsil.
  • B Mean PD-l expression (geometric MFI) across major leukocytes subsets identified in two independent non-inflamed human tonsils.
  • C Mean PD-l expression (geometric MFI) across major leukocytes subsets identified in three healthy human PBMC.
  • D An embodiment of a PD-L1 CAR design - Coding sequence for an example of a CAR construct, color-coded by sequences and separated by .
  • CAR-transduced NK-92 express surface PD-L1 - PD-L1 expression of empty- vector-transduced vs. CAR-lentivirus-transduced NK-92, including PD-L1 fluorescence-minus-one (FMO) control.
  • FMO fluorescence-minus-one
  • FIG. 2 PD-L1 CAR NK cell responses to plate- and cell-associated
  • PD-1 PD-1.
  • A Degranulation (surface exposure of CD 107 a) of control (left) or CAR (right) NK-92 following a 4-hour incubation in the presence of either anti-PD-Ll IgG vs. goat IgG control (20 pg/mL), or rhPD-l-Fc vs. IgG-Fc control (10 pg/mL). Representative data from one of three experimental replicates is shown.
  • (D) Fold degranulation (over control NK-92 with control S2) of CAR NK-92 in the presence of either control, PD-1 'A or PD-l Hl drosophila S2 cells at a 1:5 E:T for four hours (n 2-3). Dotted line represents average degranulation of control NK-92 cultured with control S2 cells. Data were analyzed via l-way ANOVA with multiple comparisons. Representative data from one of two experimental replicates is shown.
  • FIG. 3 PD-L1 CAR NK cells selectively kill TFH cells.
  • FIG. 4 PD-L1 CAR NK cells kill T cells in T:B co-cultures.
  • Control or CAR NK-92 degranulation following 4-hour co-culture with SEB-stimulated tonsillar T FH and CD27 + B cells at an NK:T:B cell ratio of 5:1:2 (n 5). Representative one of three experiments is shown. Data analyzed via Student’s t-test.
  • B Percent lysis of calcein-AM (CAM)-labeled, SEB-stimulated tonsillar T FH and CD27 + B cells after incubation with control or CAR NK-92 for four hours over an increasing E:T ratio, as measured by calcein fluorescence measured in supernatant. Shaded area is ⁇ percent background lysis of T and B cells alone.
  • E ELISA assay optical density (OD) readings at 450nm comparing levels of immunoglobulin M (IgM) in the supernatant of the aforementioned co-cultures at day 5. Dotted line represents average background readings in blank wells. *p ⁇ 0.05,
  • FIG. 5 Humanized mouse models of SLE.
  • B CD56 + ZsGreen + NK in spleen and liver 24h after injection of 10 7 CAR cells.
  • Some embodiments of the invention include chimeric polypeptides. Other embodiments of the invention include chimeric nucleic acid molecules encoding a chimeric polypeptide. Other embodiments of the invention include vectors comprising a chimeric nucleic acid molecule. Still other embodiments of the invention include cells comprising a chimeric nucleic acid molecule, a chimeric polypeptide, or both. Yet other embodiments of the invention include methods of making cells. Some embodiments include methods of treating disease. Additional embodiments of the invention are also discussed herein.
  • “wt” or“wild type” refers to a polypeptide or nucleic acid molecule that is naturally found in an organism.
  • one or more modifications of a polypeptide, a segment of a polypeptide, or a portion of a polypeptide can comprise an insertion, a deletion, a substitution, or combinations thereof.
  • one or more modifications can comprise an insertion which can comprise inserting (e.g., at the C-terminus, at the N-terminus, or at another place in the polypeptide or segment or portion thereof) about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 amino acids (e.g., natural amino acids, or modified or unusual amino acids).
  • one or more modifications can comprise a deletion which can comprise deleting (e.g., at the C-terminus, at the N-terminus, or at another place in the chimeric polypeptide or segment or portion thereof) about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 amino acids.
  • one or more modifications can comprise a substitution (e-g ⁇ , conservative substitutions) which can comprise substitutions (e.g., at the C- terminus, at the N-terminus, or at another place in the chimeric polypeptide or segment or portion thereof) for about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 amino acids (e.g., natural amino acids, or modified or unusual amino acids).
  • substitutions e.g., at the C- terminus, at the N-terminus, or at another place in the chimeric polypeptide or segment or portion thereof
  • substitutions e.g., at the C- terminus, at the N-terminus, or at another place in the chimeric polypeptide or segment or portion thereof
  • amino acids e.g., natural amino acids, or modified or unusual amino acids
  • A“portion” of a polypeptide is defined as at least 3 consecutive amino acids of that polypeptide.
  • a portion of a polypeptide can comprise at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 25, at least 30, at least 50, at least 100, at least 200, at least 500, at least 700, or at least 1000, or from about 1 to about 1000 amino acids, from about 4 to about 1000 amino acids, from about 5 to about 1000 amino acids, from about 10 to about 1000 amino acids, from about 50 to about 1000 amino acids, from about 1 to about 500 amino acids, from about 4 to about 500 amino acids, from about 5 to about 500 amino acids, from about 10 to about 500 amino acids, from about 50 to about 500 amino acids, from about 1 to about 300 amino acids, from about 4 to about 300 amino acids, from about 5 to about 300 amino acids, from about 10 to about 300 amino acids, from about 10 to
  • A“functional polypeptide” is defined as a polypeptide (e.g., a wt polypeptide or a modified polypeptide) that has desired levels (e.g., at least about 20%, at least about 40%, at least about 50%, at least about 75%, at least about 80%, at least about 90%, at least about 95%, or at least about 99%, as compared to another polypeptide, such as a naturally occurring polypeptide) of one or more functions such as, for example, the ability to bind PD-l (e.g., human PD-l, mouse PD-l, rat PD-l, monkey PD-l, mammal PD-l, primate PD-l, or SEQ ID NO:34), the ability to target TFH cells, the ability to kill TFH cells, or the ability to result in degranulation.
  • desired levels e.g., at least about 20%, at least about 40%, at least about 50%, at least about 75%, at least about 80%, at least about 90%
  • the functional polypeptide has an increased level of one or more functions as compared to another polypeptide (e.g., a naturally occurring polypeptide).
  • Nucleic acid molecules can be designed to encode for functional polypeptides, and such nucleic acid molecules are encompassed by the present invention.
  • a "functionally equivalent” polypeptide e.g., a chimeric polypeptide or a segment therein is defined as a polypeptide that has been modified (e.g., by one or more insertions, one or more deletions, or one or more substitutions (e.g., conservative substitutions)) from an original polypeptide (e.g., a wt-PD-Ll portion of a chimeric polypeptide or any portion of a chimeric polypeptide) and that modified polypeptide retains desired levels (e.g., at least about 20%, at least about 40%, at least about 50%, at least about 75%, at least about 80%, at least about 90%, at least about 95%, or at least about 99%) of one or more functions of the original polypeptide, such as, for example, the ability to bind PD-l (e.g., human PD-l, mouse PD-l, rat PD-l, monkey PD-l, mammal PD-l,
  • the shorter the length of a polypeptide the fewer the modifications (e.g., substitutions) that can be made within the polypeptide while retaining, for example, a desired level of a chosen function.
  • longer polypeptides can have a greater number of such changes while retaining, for example, a desired level of a chosen function.
  • a full-length polypeptide can have more tolerance for a fixed number of changes while retaining, for example, a desired level of a chosen function, as compared to a shorter length of that polypeptide.
  • substitutions can take many forms, including but not limited to those described herein.
  • the hydropathic index of amino acids may be considered in designing substitutions.
  • each amino acid is assigned a hydropathic index on the basis of their hydrophobicity or charge
  • threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); or arginine (-4.5).
  • certain amino acids may be substituted for other amino acids having a similar hydropathic index.
  • the substitution of amino acids with hydropathic indices can be made with amino acids that have an index difference of no more than ⁇ 2, no more than ⁇ 1, or no more than ⁇ 0.5.
  • substitutions can also be made based on
  • hydrophilicity values As detailed in U.S. Patent 4,554,101, the following hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0);
  • A“conservative substitution” in an amino acid sequence, polypeptide, or a polypeptide segment indicates that a given amino acid residue is replaced by a residue having similar physiochemical characteristics (e.g., no more than +1 when based on hydropathic index or no more than +1 when base on hydrophilicity values).
  • conservative substitutions include (a) substitution of one aliphatic residue for another with an aliphatic residue, (b) substitution of one of he, Val, Leu, or Ala for one another of Ile, Val, Leu, or Ala, (c) substitution of one of Gly, Ile, Val, Leu, or Ala for one another of Gly, he, Val, Leu, or Ala, (d) substitution of one polar residue for another polar residue, (e) substitution of one of Lys and Arg with another of Lys and Arg, (f) substitution of one of Glu and Asp with another of Glu and Asp, (g) substitution of one of Gln and Asn with another of Gln and Asn, (h) substitution of one hydroxyl or sulfur containing residue with another hydroxyl or sulfur containing residue, (i) substitution of one of Ser, Cys, Thr, or Met with another of Ser, Cys, Thr, or Met, (j) substitution of one aromatic residue for another with an aromatic residue, (k)
  • the nucleic acid molecule can be engineered to contain distinct sequences while at the same time retaining the capacity to encode a desired polypeptide. In some embodiments, this can be accomplished owing to the degeneracy of the genetic code (i.e., the presence of multiple codons) which encode for the same amino acids. In other instances, it can be accomplished by including, adding, or excluding introns in the nucleic acid molecule.
  • a restriction enzyme recognition sequence can be introduced into a nucleic acid sequence while maintaining the ability of that nucleic acid molecule to encode a desired polypeptide.
  • a CRISPR system e.g., a CRISPR system comprising one or more of guide RNA, crRNA, tracrRNA, sgRNA, DNA repair template, and Cas protein, such as but not limited to CRISPR/Cas9
  • a CRISPR system e.g., a CRISPR system comprising one or more of guide RNA, crRNA, tracrRNA, sgRNA, DNA repair template, and Cas protein, such as but not limited to CRISPR/Cas9
  • Cas protein such as but not limited to CRISPR/Cas9
  • amino acid sequences e.g., polypeptides
  • nucleic acid sequences may include additional residues, such as additional N- or C-terminal amino acids or 5' or 3' sequences, and yet still be essentially as set forth in one of the sequences disclosed herein, so long as the sequence meets the criteria set forth above, such as including the maintenance of biological activity where polypeptide expression is concerned.
  • the addition of terminal sequences particularly applies to nucleic acid sequences that may, for example, include various non-coding sequences flanking either of the 5' or 3' portions of the coding region or may include various internal sequences, (i.e., introns) which can occur within genes.
  • polypeptides in cyto, via transcription and translation of appropriate nucleic acid molecules (e.g., nucleic acid sequences as discussed herein). These polypeptides will include the twenty“natural” amino acids, and post-translational modifications thereof. In vitro peptide synthesis permits the use of modified or unusual amino acids.
  • inventive polypeptide encompasses modifications (e.g., one or more substitutions or one or more insertions) that include one or more modified or unusual amino acids.
  • modifications e.g., one or more substitutions or one or more insertions
  • a table of exemplary, but not limiting, modified or unusual amino acids is provided in Table C.
  • Some embodiments of the invention include chimeric polypeptides comprising (a) an extracellular segment, (b) a transmembrane segment, and (c) an intracellular segment.
  • the chimeric polypeptide is a single amino acid chain.
  • the extracellular segment of the chimeric polypeptide is found mostly on the outside of the cell.
  • certain portions (but not all) of the extracellular segment can bind to or be within the cell membrane.
  • none of the extracellular segment binds to the cell membrane, is within the cell membrane, or both.
  • the transmembrane segment is found mostly within the cell membrane.
  • certain portions of the transmembrane segment can be found inside the cell, outside the cell, or both. In some embodiments, the transmembrane segment is found only within the cell membrane, or none of the transmembrane segment can be found inside the cell, outside the cell, or both.
  • the intracellular segment of the chimeric polypeptide is found mostly on the inside of the cell. In some embodiments, certain portions (but not all) of the intracellular segment can bind to or be within the cell membrane. In other embodiments, none of the intracellular segment binds to the cell membrane, is within the cell membrane, or both.
  • the chimeric polypeptide is not a naturally occurring polypeptide.
  • the chimeric polypeptide is not a wt PD- Ll (e.g., not a mammalian PD-L1, a mouse PD-L1 (e.g., UniProt No. E2D891 or UniProt No. Q9EP73), a dog PD-L1 (e.g., GenBank: BA074172.1), a cat PD-L1 (e.g., UniProt No.
  • A8WEV0 a rat PD-L1, a pig PD-L1 (e.g., UniProt Q4QTK1), a woodchuck PD-L1 (e.g., UniProt B8PXK6), a cow PD-L1 (e.g., GenBank: BAH82662.1), a monkey PD-L1 (e.g., a cynomolgus monkey), a primate PD-L1, or a human PD-L1 UnitProt Q9NZQ7-1, UnitProt Q9NZQ7-2, or UnitProt Q9NZQ7-3).
  • a rat PD-L1 e.g., UniProt Q4QTK1
  • a woodchuck PD-L1 e.g., UniProt B8PXK6
  • cow PD-L1 e.g., GenBank: BAH82662.1
  • a monkey PD-L1 e.g.,
  • the chimeric polypeptide does not comprise an Fc region of an antibody (e.g., a wt antibody). In some embodiments, one or more of the extracellular segments, intracellular segments, or transmembrane segments do not comprise an Fc region of an antibody (e.g., a wt antibody).
  • the extracellular segment and the transmembrane segment are not both present in the same endogenous single-chain polypeptide in a mammal. In still other embodiments, the extracellular segment and the intracellular segment are not both present in the same endogenous single-chain polypeptide in a mammal. In yet other embodiments, the intracellular segment and the transmembrane segment are not both present in the same endogenous single-chain polypeptide in a mammal. In certain embodiments, the extracellular segment, the transmembrane segment, and the intracellular segment are not all present in the same endogenous single-chain polypeptide in a mammal.
  • the chimeric polypeptide (e.g., when in a cell, an immune cell, an NK cell, or a T cell) (a) targets follicular helper T cells (TFH), (b) kills follicular helper T cells (TFH), (C) has an extracellular segment that binds to programmed cell death protein 1 (PD-l, such as, for example, human PD-l, mouse PD-l, rat PD-l, monkey PD-l, mammal PD-l, primate PD-l, or SEQ ID NO:34), (d) results in degranulation, (e) activates the cell (e.g., an immune cell) or (f) combinations thereof.
  • PD-l programmed cell death protein 1
  • the chimeric polypeptide comprises one or more of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12,
  • SEQ ID NO: 13 SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:2l, SEQ ID NO:22, SEQ ID NO:
  • the chimeric polypeptide comprises SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:3 l, SEQ ID NO:38, combinations thereof, or one or more modifications thereof.
  • the chimeric polypeptide comprises SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:3 l, or one or more modifications thereof.
  • the extracellular segment is linked to the transmembrane segment by a linker with one or more amino acids (e.g., about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 15, about 20, about 25, about 50, about 100, about 200, or about 500 amino acids, or from about 1 to about 50, from about 1 to about 200, or from about 1 to about 500).
  • one or more amino acids e.g., about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 15, about 20, about 25, about 50, about 100, about 200, or about 500 amino acids, or from about 1 to about 50, from about 1 to about 200, or from about 1 to about 500.
  • the transmembrane segment is linked to the intracellular segment by a linker with one or more amino acids (e.g., about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 15, about 20, about 25, about 50, about 100, about 200, or about 500 amino acids, or from about 1 to about 50, from about 1 to about 200, or from about 1 to about 500).
  • amino acids e.g., about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 15, about 20, about 25, about 50, about 100, about 200, or about 500 amino acids, or from about 1 to about 50, from about 1 to about 200, or from about 1 to about 500).
  • the extracellular segment comprises wild type programmed cell death ligand 1 (wt-PD-Ll), wt-PD-Ll with one or more modifications, or a portion of wt-PD- Ll with optionally one or more modifications.
  • PD-L1 is also known as CD274 and B7- Hl.
  • the wt-PD-Ll includes any full length naturally occurring PD-L1 (i.e., wt PD-L1), such as, but not limited to, a mammalian PD-L1, a mouse PD-L1 (e.g., UniProt No.
  • E2D891 or UniProt No. Q9EP73 a dog PD-L1 (e.g., GenBank: BA074172.1), a cat PD-L1 (e.g., UniProt No. A8WEV0), a rat PD-L1, a pig PD-L1 (e.g., UniProt Q4QTK1), a woodchuck PD-L1 (e.g., UniProt B8PXK6), a cow PD-L1 (e.g., GenBank:
  • a monkey PD-L1 e.g., a cynomolgus monkey
  • a primate PD-L1 e.g., a human PD-L1
  • SEQ ID NO:38 UnitProt Q9NZQ7-1
  • UnitProt Q9NZQ7-2 UnitProt Q9NZQ7-3
  • the extracellular segment comprises the amino acid sequence ADYKR (SEQ ID NO: 14) or ADYK (SEQ ID NO: 15).
  • the extracellular segment comprises about 3, about 4, about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 50, about 100, about 150, about 200, about 250, about 350, about 400, about 450, about 500, about 750, or about 1000 amino acids, or about 2000 amino acids, or from about 3 to about 2000 amino acids, from about 4 to about 2000 amino acids, from about 5 to about 2000 amino acids, from about 10 to about 2000 amino acids, from about 50 to about 2000 amino acids, from about 3 to about 1000 amino acids, from about 4 to about 1000 amino acids, from about 5 to about 1000 amino acids, from about 10 to about 1000 amino acids, from about 50 to about 1000 amino acids, from about 3 to about 500 amino acids, from about 4 to about 500 amino acids, from about 5 to about 500 amino acids, from about 10 to about 500 amino acids, from about 50 to about 500 amino acids, from about 3 to to
  • the Kd (dissociation constant) of the extracellular segment (or of the chimeric polypeptide) with PD-l is about 100 nm, about 200 nm, about 300 nm, about 400 nm, about 500 nm, about 750 nm, about 1000 nm, about 2000 nm, about 3000 nm, about 4000 nm, about 5000 nm, about 7000 nm, about 8000 nm, about 9000 nm, about
  • PD-L1 in some instances, can include an insertion, a deletion, a substitution, or combinations thereof.
  • one or more modifications to a wt-PD-Ll can comprise an insertion which can comprise inserting (e.g., at the C-terminus, at the N- terminus, or at another place in the polypeptide or segment or portion thereof) about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 amino acids (e.g., natural amino acids, or modified or unusual amino acids).
  • one or more modifications to a wt-PD-Ll can comprise a deletion which can comprise deleting (e.g., at the C-terminus, at the N-terminus, or at another place in the chimeric polypeptide or segment or portion thereof) about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 amino acids.
  • one or more modifications to a wt-PD-Ll can comprise a substitution (e.g., conservative substitutions) which can comprise substitutions (e.g., at the C-terminus, at the N-terminus, or at another place in the chimeric polypeptide or segment or portion thereof) for about 1, about 2, about 3, about 4, about 5, about 6, about
  • a substitution e.g., conservative substitutions
  • substitutions e.g., at the C-terminus, at the N-terminus, or at another place in the chimeric polypeptide or segment or portion thereof
  • the modified PD-L1 results in a chimeric polypeptide that is a functional PD-L1 polypeptide (e.g., the ability to bind PD-l (e.g., human PD-l, mouse PD-l, rat PD-l, monkey PD-l, mammal PD-l, primate PD-l, or SEQ ID NO:34 - see also ZAK et al.
  • PD-l e.g., human PD-l, mouse PD-l, rat PD-l, monkey PD-l, mammal PD-l, primate PD-l, or SEQ ID NO:34 - see also ZAK et al.
  • the modified PD-L1 results in a chimeric polypeptide that is functionally equivalent to a wt-PD-Ll polypeptide with regard to, for example, the ability to bind PD-l (e.g., human PD-l, mouse PD-l, rat PD-l, monkey PD- 1, mammal PD-l, primate PD-l, or SEQ ID NO:34), the ability to target TFH cells, the ability to kill TFH cells, or the ability to result in degranulation.
  • PD-l e.g., human PD-l, mouse PD-l, rat PD-l, monkey PD- 1, mammal PD-l, primate PD-l, or SEQ ID NO:34
  • the extracellular segment does not encompass one or more naturally occurring polypeptides (e.g., does not encompass one or more of the wt-PD-Lls, e.g., human PD-l, mouse PD-l, rat PD-l, monkey PD-l, mammal PD-l, primate PD-l, or SEQ ID NO:34). In other embodiments, the extracellular segment does not encompass any of the wt-PD-Lls (e.g., human PD-l, mouse PD-l, rat PD-l, monkey PD-l, mammal PD-l, primate PD-l, or SEQ ID NO:34). In some embodiments, the extracellular segment does not encompass any naturally occurring polypeptide (e.g., does not encompass any of the wt-PD-Ll or any other naturally occurring polypeptide).
  • the extracellular segment does not encompass any naturally occurring polypeptide (e.g., does not encompass any of the wt-PD-Ll
  • one or more modifications to a wt-PD-Ll can include one or more substitutions, one or more insertions, or one or more deletions (or combinations thereof) to one or more amino acids in a hydrophobic region of a wt-PD- Ll, in a signal region of a wt-PD-Ll, in a transmembrane region of a wt-PD-Ll, or in a combination thereof.
  • one or more modifications to a wt-PD-Ll can include one or more substitutions or one or more deletions (or combinations thereof) to one or more amino acids in a hydrophobic region of a wt-PD-Ll, in a signal region of a wt-PD-Ll, in a transmembrane region of a wt-PD-Ll, or in a combination thereof.
  • the extracellular segment can comprise a polypeptide sequence with an amino acid sequence identity to a wt-PD-Ll (e.g., a mammalian PD-L1, a mouse PD-L1 (e.g., UniProt No. E2D891 or UniProt No. Q9EP73), a dog PD-L1 (e.g., GenBank: BA074172.1), a cat PD-L1 (e.g., UniProt No.
  • a wt-PD-Ll e.g., a mammalian PD-L1, a mouse PD-L1 (e.g., UniProt No. E2D891 or UniProt No. Q9EP73)
  • a dog PD-L1 e.g., GenBank: BA074172.1
  • a cat PD-L1 e.g., UniProt No.
  • A8WEV0 a rat PD-L1, a pig PD-L1 (e.g., UniProt Q4QTK1), a woodchuck PD-L1 (e.g., UniProt B8PXK6), a cow PD-L1 (e.g., GenBank: BAH82662.1), a monkey PD-L1 (e.g., a cynomolgus monkey), a primate PD-L1, or a human PD-L1 (e.g., SEQ ID
  • the extracellular segment sequence has an amino acid sequence identity to a mammalian PD-L1, a mouse PD-L1 (e.g., UniProt No. E2D891 or UniProt No. Q9EP73 ), a dog PD-L1 (e.g., GenBank: BA074172.1), a cat PD-L1 (e.g., UniProt No. A8WEV0), a rat PD-L1, a pig PD-L1 (e.g., UniProt Q4QTK1), a woodchuck PD-L1 (e.g., UniProt B8PXK6), a cow PD-L1 (e.g., GenBank:
  • a monkey PD-L1 e.g., a cynomolgus monkey
  • a primate PD-L1 e.g., SEQ ID NO:38 (UnitProt Q9NZQ7-1), UnitProt Q9NZQ7-2, or UnitProt Q9NZQ7-3) of about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.1%, about 99.2%, about 99.3%, about 99.4%, about 99.5%, about 99.6%, about 99.7%, about 99.8%, about 99.9%, about 99.95%, about 99.99%, less than about 100%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 99%, or at least about 99.5%.
  • SEQ ID NO:38 UnitProt Q9NZQ7-1
  • amino acid sequence identity can be determined by any suitable method, such as using BLAST, BLAST-2, ALIGN, ALIGN-2, Clustal Omega, or Megalign software. Unless otherwise indicated, the amino acid sequence identity (e.g., percent identity) is determined using BLAST-2.
  • the extracellular segment optionally comprises one or more (e.g., 1, 2, 3, 4, or 5) spacer sequences.
  • one or more of the spacer sequences (a) are between the PD-L1 portion (e.g., wt-PD-Ll or a
  • each of the one or more spacer sequences can comprise up to about 1000 amino acids (e.g., about 1, about 5, about 10, about 20, about 25, about 50, about 100, about 150, about 200, about 250, about 300 amino acids, about 500 amino acids, or about 1000 amino acids, from 5 to about 25, from 5 to about 50, from 5 to about 100, from 5 to about 300, from 5 to about 500, from 5 to about 1000 amino acids, or 1 to about 1000 amino acids).
  • one or more spacer sequences can be a polypeptide (e.g., from an animal, mammal, mouse, rat, primate, monkey, or human) comprising variable regions of an antibody (e.g.
  • TCR T cell receptor
  • CD4 e.g., TCRa, TCRp, TCRy, or TCR5
  • CD8a CD8p
  • CD49A, CD49B, CD49D, CD49E, CD49F CD61, CD41, or CD51, or one or more modifications thereof, or combinations thereof.
  • one or more spacer sequences can be a polypeptide (e.g., from an animal, mammal, mouse, rat, primate, monkey, or human) comprising or consisting of CD8a, a portion of CD8a (e.g., a hinge sequence or a leader sequence),
  • the spacer sequence can be a polypeptide comprising SEQ ID NO.: 13, amino acid numbers 118 to 178 of CD8a (NCBI Ref Seq: NP_00l759.3) which is a hinge sequence of CD8a, CD8a leader sequence (e.g., SEQ ID NO: 10), amino acid numbers 135 to 195 of CD8P
  • the one or more spacer sequences may be an artificially synthesized sequence.
  • the extracellular segment comprises one or more of
  • SEQ ID NO.l SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.
  • the transmembrane segment can comprise a wt polypeptide, a modification of a wt polypeptide, or an artificially designed polypeptide.
  • the transmembrane segment can be a polypeptide portion (or modification thereof) obtained from any membrane-binding or transmembrane protein or polypeptide.
  • the transmembrane segment can comprise of a T cell receptor a or b chain, a O ⁇ 3z chain, CD28, CD3e, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, Inducible T-cell COStimulator (ICOS), CD154, or a glucocorticoid-induced TNFR-related protein (GITR), or portions, or one or more modifications thereof.
  • the artificially designed transmembrane segment can be a polypeptide comprising hydrophobic residues such as leucine and valine.
  • a triplet of phenylalanine, tryptophan and valine can be found at each end of the synthetic transmembrane segment.
  • the transmembrane segment comprises about 3, about 4, about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 50, about 100, about 150, about 200, about 250, about 350, about 400, about 450, about 500, about 750, or about 1000 amino acids, or from about 3 to about 1000 amino acids, from about 4 to about 1000 amino acids, from about 5 to about 1000 amino acids, from about 10 to about 1000 amino acids, from about 50 to about 1000 amino acids, from about 3 to about 500 amino acids, from about 4 to about 500 amino acids, from about 5 to about 500 amino acids, from about 10 to about 500 amino acids, from about 50 to about 500 amino acids, from about 3 to about 300 amino acids, from about 4 to about 300 amino acids, from about 5 to about 300 amino acids, from about 10 to about 300 amino acids, or from about 50 to about 300 amino acids.
  • a linker e.g., a linker having a length of about 1 to about 10 amino acids, of about 1 to about 25 amino acids, of about 1 to about 50 amino acids, or of about 1 to about 100 amino acids
  • the transmembrane segment can be SEQ ID NO: 16, SEQ ID NO: 17, the sequence of amino acid numbers 153 to 179 of CD28 (NCBI Ref Seq: NP_006l30.l), or portions thereof, or one or more modifications thereof.
  • the intracellular segment can comprise one or more polypeptides to transmit a signal into a cell (e.g., immune cell, NK cells or T Cells) when the extracellular segment of the chimeric polypeptide interacts with (e.g., binds to) another molecule (e.g., on another cell).
  • a cell e.g., immune cell, NK cells or T Cells
  • another molecule e.g., on another cell.
  • a signal can be any suitable signal, including but not limited to phosphorylation or de-phosphorylation, a signal that results in phosphorylation or de-phosphorylation, association or dissociation of intracellular molecules, a signal that results in association or dissociation of intracellular molecules, a signal that results in increasing or decreasing intracellular ion concentrations (e.g., Ca), a signal that results in increasing or decreasing gene expression, a signal that results in degranulation, or any other signaling activity found in immune cells (e.g., NK cells or T Cells).
  • the intracellular portions (or modifications thereof) from other polypeptides e.g., signal transmitting polypeptides
  • Any suitable signal transmitting polypeptide can be used including but not limited to cytoplasmic sequences from a TCR complex or a costimulatory molecule, or portions thereof, or one or more modifications thereof (e.g., to be functionally
  • the intracellular segment comprises about 3, about 4, about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 50, about
  • amino acids 100, about 150, about 200, about 250, about 350, about 400, about 450, about 500, about 750, or about 1000 amino acids, or about 2000 amino acids, or from about 3 to about 2000 amino acids, from about 4 to about 2000 amino acids, from about 5 to about 2000 amino acids, from about 10 to about 2000 amino acids, from about 50 to about 2000 amino acids, from about 3 to about 1000 amino acids, from about 4 to about 1000 amino acids, from about 5 to about 1000 amino acids, from about 10 to about 1000 amino acids, from about 50 to about 1000 amino acids, from about 3 to about 500 amino acids, from about 4 to about 500 amino acids, from about 5 to about 500 amino acids, from about 10 to about 500 amino acids, from about 50 to about 500 amino acids, from about 3 to about 300 amino acids, from about 4 to about 300 amino acids, from about 5 to about 300 amino acids, from about 10 to about 300 amino acids, or from about 50 to about 300 amino acids.
  • the intracellular segment of the chimeric polypeptide can comprise a first signaling polypeptide and optionally one or more second signaling polypeptides.
  • the signaling of the first signaling polypeptide can be dependent on the extracellular segment interacting (e.g., binding) with another molecule.
  • the signaling of the one or more second signaling polypeptides is not directly dependent on the extracellular segment interacting (e.g., binding) with another molecule.
  • the first signaling polypeptide comprises about 3, about 4, about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 50, about 100, about 150, about 200, about 250, about 350, about 400, about 450, about 500, about 750, or about 1000 amino acids, or about 2000 amino acids, or from about 3 to about 2000 amino acids, from about 4 to about 2000 amino acids, from about 5 to about 2000 amino acids, from about 10 to about 2000 amino acids, from about 50 to about 2000 amino acids, from about 3 to about 1000 amino acids, from about 4 to about 1000 amino acids, from about 5 to about 1000 amino acids, from about 10 to about 1000 amino acids, from about 50 to about 1000 amino acids, from about 3 to about 500 amino acids, from about 4 to about 500 amino acids, from about 5 to about 500 amino acids, from about 10 to about 500 amino acids, from about 50 to about 500 amino acids, from about 3 to about 300 amino acids, from about 4 to about 300 amino acids, from about 5 to about 300 amino acids, from about 10 to about 300 amino acids, or from about
  • one of the optional second signaling polypeptide comprises about 3, about 4, about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 50, about 100, about 150, about 200, about 250, about 350, about 400, about 450, about 500, about 750, or about 1000 amino acids, or about 1500 amino acids, or from about 3 to about 1500 amino acids, from about 4 to about 2000 amino acids, from about 5 to about 1500 amino acids, from about 10 to about 1500 amino acids, from about 50 to about 1500 amino acids, from about 3 to about 1000 amino acids, from about 4 to about 1000 amino acids, from about 5 to about 1000 amino acids, from about 10 to about 1000 amino acids, from about 50 to about 1000 amino acids, from about 3 to about 500 amino acids, from about 4 to about 500 amino acids, from about 5 to about 500 amino acids, from about 10 to about 500 amino acids, from about 50 to about 500 amino acids, from about 3 to about 300 amino acids, from about 4 to about 300 amino acids, from about 5 to about 300 amino acids, from about 10 to about 300 amino acids, or from about 50
  • the first signaling polypeptide can be any suitable polypeptide that can stimulate (directly or indirectly) the activation of an immune cell (e.g., an NK cell or a T cell).
  • the first signaling polypeptide can comprise a signal transduction motif known as an immunoreceptor tyrosine-based activation motif (IT AM) (e.g., two Tyr-x-x-Leu/Ile elements (where x is any amino acid) separated by six to eight amino acid residues) (e.g., see, RETH (1989)“Antigen receptor tail clue” Nature, Vol. 338, pp. 383-384, which is incorporated herein by reference in its entirety).
  • IT AM immunoreceptor tyrosine-based activation motif
  • the first signaling polypeptide can comprise a signal transduction motif known as an immunoreceptor tyrosine-based inhibition motif (ITIM) (e.g., Ile/Val/Leu/Ser-x-Tyr-x-x-Leu/Val, where x denotes any amino acid) (e.g., see BURSHTYN et al. (1999)“Conserved Residues Amino-Terminal of Cytoplasmic Tyrosines Contribute to the SHP-l -Mediated Inhibitory Function of Killer Cell Ig-Like Receptors” J Immunol., Vol. 162, No. 2, pp. 897-902, which is incorporated herein by reference in its entirety).
  • ITIM immunoreceptor tyrosine-based inhibition motif
  • the first signaling polypeptide can comprise CD3z,
  • the first signaling polypeptide can comprise SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, amino acid numbers 51 to 164 of O ⁇ 3z (NCBI Ref Seq: NP 932170.1), amino acid numbers 45 to 86 of FceRfy (NCBI Ref Seq: NP 004097.1), amino acid numbers 201 to 244 of FceRip (NCBI Ref Seq: NP 000130.1), amino acid numbers 139 to 182 of CD3y (NCBI Ref Seq: NP _000064.l), amino acid numbers 128 to 171 ofCD35 (NCBI Ref Seq: NP_000723.l), amino acid numbers 153 to 207 of CD3e (NCBI Ref Seq: NP 000724.1 ), amino acid numbers 402
  • the second signaling polypeptide can comprise
  • the second signaling polypeptide can comprise SEQ ID NO: 21, SEQ ID NO: 22, amino acid numbers 236 to 351 of CD2 (NCBI Ref Seq: NP 001758.2), amino acid numbers 421 to 458 of CD4 (NCBI Ref Seq: NP 000607.1), amino acid numbers 402 to 495 of CD5 (NCBI Ref Seq: NP 055022.2), amino acid numbers 207 to 235 of CD8a (NCBI Ref Seq: NP 001759.3), amino acid numbers 196 to 210 of CD8P (GenBank: AAA35664.1), amino acid numbers 181 to 220 of CD28 (NCBI Ref Seq: NP 006130.1), amino acid numbers 214 to 255 of CD137 (4-1BB, NCBI Ref Seq: NP_00l552.2
  • linkers (which can be the same or different) can be between the first signaling polypeptide and a second signaling polypeptide or between two second signaling polypeptides.
  • the linkers can be, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, or 100 amino acids (e.g., from 2 to 10 amino acids).
  • a linker having a glycine-serine continuous sequence can be used.
  • the intracellular segment comprises SEQ ID NO:
  • SEQ ID NO: 19 SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, or one or more modifications thereof.
  • the chimeric polypeptide comprises SEQ ID NO:
  • nucleic acid molecules that can encode for the chimeric polypeptide (“chimeric nucleic acid molecules”).
  • the chimeric nucleic acid molecule is included in a vector (e.g., a viral vector, a retroviral vector, a lentiviral vector, an adenoviral vector, an adeno-associated viral vector, a herpesviral vector, a chimeric viral vector, a plasmid, a cosmid, an artificial chromosome, a bacteriophage, an animal virus, a plant virus, an expression vector, a conjugative vector, a nonconjugative vector or an mRNA transfection).
  • a vector e.g., a viral vector, a retroviral vector, a lentiviral vector, an adenoviral vector, an adeno-associated viral vector, a herpesviral vector, a chimeric viral vector, a plasmid, a cosmid, an artificial chromosome,
  • the chimeric nucleic acid molecule is in a cell, such as an insect cell (e.g., an Sf9 cell) or a mammalian cell (e.g., a human cell, a rat cell, a mouse cell, a CHO cell, an immune cell, an immune cell progenitor, a T cell, a T cell progenitor, an NK cell, KHYG1, NK-92, YT, SNK-6, NKL, or an NK cell progenitor).
  • the chimeric nucleic acid molecule is in a cell and the cell is isolated from an animal, a mammal, a primate, or a human.
  • the chimeric nucleic acid molecule comprises one or more nucleic acid sequences that are not used to encode for the chimeric polypeptide (e.g., one or more introns).
  • the chimeric nucleic acid molecule can comprise a nucleic acid sequence as found in nature (e.g., including introns).
  • the chimeric nucleic acid molecule differs from the one or more nucleic acid molecules in nature because the chimeric nucleic acid molecule does not include one or more introns.
  • the chimeric nucleic acid molecule is a cDNA molecule (“chimeric cDNA molecule”).
  • the segments of the chimeric cDNA molecule are identical to a nucleic acid molecule found in nature. In other embodiments, the segments of the chimeric cDNA molecule are not identical to a nucleic acid molecule found in nature (e.g., due to a segment of the cDNA molecule not including one or more introns in the nucleic acid molecule found in nature).
  • the chimeric nucleic acid molecule sequence has a sequence identity to a nucleic acid molecule encoding a chimeric polypeptide (e.g., SEQ ID NO:32) of about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.1%, about 99.2%, about 99.3%, about 99.4%, about 99.5%, about 99.6%, about 99.7%, about 99.8%, about 99.9%, about 99.95%, about 99.99%, less than about 100%, at least about 90%, at least about 95%, at least about 99%, or at least about 99.5%.
  • a chimeric polypeptide e.g., SEQ ID NO:32
  • the chimeric nucleic acid molecule sequence has a sequence identity to SEQ ID NO:32 of about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.1%, about 99.2%, about 99.3%, about 99.4%, about 99.5%, about 99.6%, about 99.7%, about 99.8%, about 99.9%, about 99.95%, about 99.99%, less than about 100%, at least about 90%, at least about 95%, at least about 99%, or at least about 99.5%.
  • nucleic acid sequence identity can be determined by any suitable method, such as using BLAST, BLAST-2, ALIGN, ALIGN-2, Clustal Omega, or Megalign software. Unless otherwise indicated, the nucleic acid sequence identity (e.g., percent identity) is determined using BLAST-2.
  • the chimeric nucleic acid molecule can be made using any suitable technique, such as but not limited to, chemical synthesis, enzymatic production or biological production.
  • Chemical synthesis of a nucleic acid molecule can include, for example, a nucleic acid molecule made by in vitro chemical synthesis using
  • Enzymatically produced nucleic acid molecules can be accomplished using any suitable method including but not limited to
  • Biologically produced nucleic acid molecules can be accomplished using any suitable method including but not limited to a recombinant nucleic acid produced (i.e., replicated) in a living cell, such as a recombinant DNA vector replicated in bacteria.
  • nucleic acid molecules and/or polypeptides of the present invention are encompassed within some embodiments of the present invention.
  • a polypeptide can be modified (e.g., by one or more insertions, one or more deletions, or one or more substitutions (e.g., conservative substitutions)).
  • the polypeptide which was modified does not have an appreciable loss (e.g., a decrease in a function of less than about 1%, less than about 5%, less than about 10%, less than about 25%, less than about 50%, less than about 75%, less than about 90%, less than about 95%, less than about 99%, or less than about 100%) of one or more chosen functions of the unmodified polypeptide such as, for example, targeting TFH, killing TFH, binding to PD-l (e.g., human PD-l, mouse PD-l, rat PD-l, monkey PD-l, mammal PD-l, primate PD-l, or SEQ ID NO:34), or inducing degranulation.
  • an appreciable loss e.g., a decrease in a function of less than about 1%, less than about 5%, less than about 10%, less than about 25%, less than about 50%, less than about 75%, less than about 90%, less than about 95%, less than about 99%,
  • the polypeptide which was modified retains desired levels (e.g., at least about 20%, at least about 40%, at least about 50%, at least about 75%, at least about 80%, at least about 90%, at least about 95%, or at least about 99%) of one or more functions of the unmodified polypeptide, such as, for example, targeting TFH, killing TFH, binding to PD-l (e.g., human PD-l, mouse PD-l, rat PD-l, monkey PD-l, mammal PD-l, primate PD-l, or SEQ ID NO:34), or inducing degranulation.
  • the polypeptide after modification has an increased level of one or more functions as compared to the unmodified polypeptide.
  • Nucleic acid molecules can be designed to encode for such a modified polypeptide, and such nucleic acid molecules are encompassed by the present invention.
  • the presently disclosed subject matter further includes a method of producing a chimeric polypeptide.
  • Any suitable method can used to make the chimeric polypeptides including but not limited to expression through any suitable molecular biological technique (e.g., using a prokaryotic or eukaryotic expression system), isolation from a source in nature, or chemical synthesis.
  • Eukaryotic expression systems include plant-based systems; insect cell systems via recombinant baculoviruses; whole insect systems via recombinant baculoviruses; genetically engineered yeast systems, including but not limited to Saccharomyces sp. and Picchia spp.
  • useful plant-based expression systems can include transgenic plant systems.
  • useful plant-based expression systems can include transplastomic plant systems.
  • a method of producing the chimeric polypeptide includes providing a host cell comprising a nucleic acid molecule, as disclosed herein, operatively linked to a promoter operable under conditions whereby the encoded polypeptide is expressed; and recovering the polypeptide from the host cell.
  • Some embodiments of the invention include cells comprising a chimeric polypeptide, a chimeric nucleic acid molecule or both.
  • Adding (e.g., transfection, transformation, or transduction) a chimeric nucleic acid molecule to a cell can be accomplished using any suitable method including but not limited to one or more of transformation (as used herein transfection methods are encompassed by the term transformation), viral transformation (e.g., using a viral vector, a retroviral vector, a lentiviral vector, an adenoviral vector, an adeno-associated viral vector, a herpesviral vector, a chimeric viral vector, a plasmid, a cosmid, an artificial chromosome, a bacteriophage, a virus, an animal virus, a plant virus, an expression vector, a conjugative vector, a nonconjugative vector or an mRNA transfection), injection, microinjection, electroporation, sonication, calcium ion treatment, calcium phosphate precipitation, PEG-DMSO treatment, DE-Dextran treatment, liposome mediated transformation, or a
  • transformation
  • Adding a chimeric polypeptide to modify a cell can be accomplished using any suitable method including but not limited to one or more of injection, microinjection, electroporation, sonication, calcium ion treatment, calcium phosphate precipitation, PEG-DMSO treatment, DE- Dextran treatment, or liposome mediated.
  • the added nucleic acid molecule can be part of a vector (e.g., a viral vector, a retroviral vector, a lentiviral vector, an adenoviral vector, an adeno-associated viral vector, a herpesviral vector, a chimeric viral vector, a plasmid, a cosmid, an artificial chromosome, a bacteriophage, an animal virus, a plant virus, an expression vector, a conjugative vector, a nonconjugative vector, or an mRNA transfection), a plasmid, a cosmid, an artificial chromosome, a bacteriophage, a virus, an animal virus, or a plant virus.
  • a vector e.g., a viral vector, a retroviral vector, a lentiviral vector, an adenoviral vector, an adeno-associated viral vector, a herpesviral vector, a chimeric viral vector, a plasmid
  • the added nucleic acid molecule is exogenous;“exogenous” means (a) that the added nucleic acid molecule originates from outside of the cell (e.g., is foreign to the cell).
  • the added polypeptide is exogenous;“exogenous” in this context means that the added polypeptide originates from outside of the cell (e.g., is foreign to the cell).
  • the cell can be any suitable cell including but not limited to an insect cell
  • an Sf9 cell e.g., an Sf9 cell
  • a vertebrate cell e.g., an Sf9 cell
  • a mammalian cell e.g., a human cell, a rat cell a mouse cell, a muscle cell, a non-muscle cell, a myoblast, a fibroblast, a C2C12 cell, a 10T 1 ⁇ 2 fibroblast, a NIH/3T3 cell, a CHO cell, a mesenchymal stem cell (MSC), a hematopoietic stem cell, a blood cell, a bone marrow cell, a stem cell, an adipose stem cell, an immune cell, a T cell, or an NK cell (e.g., KHYG1, NK-92, YT, SNK-6, or NKL cells)).
  • MSC mesenchymal stem cell
  • a hematopoietic stem cell e.g., KHY
  • an cell can be any suitable cell including but not limited insect cell, a vertebrate cell, or a mammalian cell (e.g., a human cell, a rat cell a mouse cell, a NIH/3T3 cell, a CHO cell, a mesenchymal stem cell (MSC), a hematopoietic stem cell, a blood cell, a bone marrow cell, a stem cell, an adipose stem cell, an immune cell, a T cell, or an NK cell (e.g., KHYG1, NK-92, YT, SNK-6, or NKL cells)).
  • a mammalian cell e.g., a human cell, a rat cell a mouse cell, a NIH/3T3 cell, a CHO cell, a mesenchymal stem cell (MSC), a hematopoietic stem cell, a blood cell, a bone marrow cell, a stem cell, an
  • the cell is autologous, allogeneic, a peripheral blood NK cell, a cord blood (allogeneic) NK cell, induced pluripotent stem cells, or an iPSC-derived NK cells (autologous or allogeneic).
  • a process for producing a cell comprising a chimeric polypeptide, a chimeric nucleic acid molecule or both can comprise a step of introducing a chimeric nucleic acid molecule into a cell using any method disclosed herein.
  • the step can be carried out ex vivo.
  • a cell can be transformed ex vivo with a virus vector or a non virus vector carrying a chimeric nucleic acid molecule to produce a cell expressing a chimeric polypeptide.
  • the cell can be a cell from a mammal, a primate, a monkey, a human, a mouse, a rat, a pig, a horse, or a dog.
  • the cell used in the process of the present disclosure is not particularly limited, and any suitable cell can be used.
  • a cell can be collected, isolated, and/or purified from a body fluid, a tissue or an organ such as blood (e.g., peripheral blood or umbilical cord blood) or bone marrow or a cell obtained by differentiating or reprogramming the aforementioned cell to produce an induced pluripotent stem cell (iPSC) can be used.
  • a tissue or an organ such as blood (e.g., peripheral blood or umbilical cord blood) or bone marrow
  • a cell obtained by differentiating or reprogramming the aforementioned cell to produce an induced pluripotent stem cell (iPSC) can be used.
  • iPSC induced pluripotent stem cell
  • a suitable cell including but not limited to a peripheral blood mononuclear cell (PBMC), an immune cell (e.g., a T cell, a dendritic cell, a B cell, a hematopoietic stem cell, a macrophage, a monocyte, a NK cell (e.g., KHYG1, NK-92, YT, SNK-6, or NKL cells) or a hematopoietic cell (e.g., a neutrophil or a basophil) , an umbilical cord blood mononuclear cell, a fibroblast, a precursor adipocyte, a hepatocyte, a skin keratinocyte, a mesenchymal stem cell, an adipose stem cell, various cancer cell strains, or a neural stem cell can be used.
  • PBMC peripheral blood mononuclear cell
  • an immune cell e.g., a T cell, a dendritic cell, a
  • an NK cell e.g., KHYG1, NK-92, YT, SNK-6, or NKL cells
  • a T cell e.g., a T cell, a precursor cell of a T cell (e.g., a hematopoietic stem cell or a lymphocyte precursor cell) or a cell population containing them
  • Any suitable T cell can be used including but not limited a CD8-positive T cell, a CD4-positive T cell, a regulatory T cell, a cytotoxic T cell, and a tumor infiltrating lymphocyte.
  • Any suitable NK cell can be used including but not limited to KHYG1, NK-92, YT, SNK-6, or NKL cells.
  • the aforementioned cells may be collected from an animal (e.g., mammal, primate, or human), obtained by expansion culture of a cell collected from an animal
  • the chimeric nucleic acid molecule can be introduced into a cell collected from the an animal (e.g., mammal, primate, or human) itself or a conspecific an animal (e.g., mammal, primate, or human) thereof.
  • the chimeric nucleic acid molecule can be inserted into a vector (e.g., as disclosed herein), and the vector can be introduced into a cell.
  • a virus vector such as a retrovirus vector (including an oncoretro virus vector, a lentivirus vector, and a pseudotyped vector), an adenovirus vector, an adeno-associated virus (AAV) vector, a simian virus vector, a vaccinia virus vector or a sendai virus vector, an Epstein - Barr virus (EBV) vector, a HSV vector, or an mRNA transfection can be used.
  • a virus vector lacking the replicating ability so as not to self-replicate in an infected cell can be used.
  • a non-virus vector can also be used in the present disclosure in combination with a liposome or a condensing agent such as a cationic lipid as described in WO 96/10038 (which is incorporated herein by reference in its entirety), WO 97/18185 (which is incorporated herein by reference in its entirety), WO 97/25329 (which is incorporated herein by reference in its entirety), WO 97/30170 (which is incorporated herein by reference in its entirety), and WO 97/31934 (which is incorporated herein by reference in its entirety).
  • the chimeric nucleic acid molecule can, in some embodiments, be also introduced into a cell by calcium phosphate transduction, DEAE-dextran, electroporation, or particle bombardment.
  • the process of the present disclosure can be carried out by selecting a suitable packaging cell based on an LTR sequence and a packaging signal sequence possessed by the vector and preparing a retrovirus particle using the packaging cell.
  • the packaging cell include PG13 (ATCC CRL 10686), PA317 (ATCC CRL-9078), GP+E-86 and GP+envAm-l2 (US Pat. No. 5,278,056, which is incorporated herein by reference in its entirety), and Psi-Crip.
  • a retrovirus particle can also be prepared using a 293 cell or a 293T cell having high transfection efficiency.
  • a method of producing a cell disclosed herein comprises adding a chimeric nucleic acid molecule or a vector described herein, to a cell.
  • a method of producing a cell herein disclosed e.g., a cell comprising a chimeric
  • polypeptide a chimeric nucleic acid molecule or both, such as, for example, a cell expressing the chimeric polypeptide
  • a) isolating a first cell e.g., a cell disclosed here, such as, for example, an immune cell, T cell or NK cells (e.g., KHYG1, NK-92, YT, SNK-6, or NKL cells) from an animal (e.g., mammal, primate, or human));
  • a first cell e.g., a cell disclosed here, such as, for example, an immune cell, T cell or NK cells (e.g., KHYG1, NK-92, YT, SNK-6, or NKL cells) from an animal (e.g., mammal, primate, or human));
  • a chimeric nucleic acid molecule e.g., contained within a vector as disclosed herein
  • a second cell e.g., a cell comprising a chimeric polypeptide, a chimeric nucleic acid molecule or both, such as, for example, a cell expressing the chimeric polypeptide
  • the first cell is an insect cell (e.g., an Sf9 cell), a vertebrate cell, or a mammalian cell (e.g., a human cell, a rat cell a mouse cell, a muscle cell, a non-muscle cell, a myoblast, a fibroblast, a C2C12 cell, a 10T 1 ⁇ 2 fibroblast, a NIH/3T3 cell, a CHO cell, a mesenchymal stem cell (MSC), a hematopoietic stem cell, a blood cell, a bone marrow cell, a stem cell, an adipose stem cell, an immune cell, a T cell, or an NK cell (e.g., KHYG1, NK-92, YT, SNK-6, or NKL cells)).
  • a mammalian cell e.g., a human cell, a rat cell a mouse cell, a muscle cell, a non-muscle cell
  • the first cell can be any suitable cell including but not limited insect cell, a vertebrate cell, or a mammalian cell (e.g., a human cell, a rat cell a mouse cell, a
  • the first cell is autologous, allogeneic, a peripheral blood NK cell, a cord blood
  • the first cell can be a cell from a mammal, a primate, a monkey, a human, a mouse, a rat, a pig, a horse, or a dog.
  • the first cell can be collected, isolated, and/or purified from a body fluid, a tissue or an organ such as blood (e.g., peripheral blood or umbilical cord blood) or bone marrow or a cell obtained by differentiating or reprogramming the
  • aforementioned cell to produce an induce pluripotent stem cell can be used.
  • Any suitable cell can used for the first cell including but not limited to a peripheral blood mononuclear cell (PBMC), an immune cell (e.g., a T cell, a dendritic cell, a B cell, a hematopoietic stem cell, a macrophage, a monocyte, a NK cell (e.g., KHYG1, NK-92, YT, SNK-6, or NKL cells) or a hematopoietic cell (e.g., a neutrophil or a basophil) , an umbilical cord blood mononuclear cell, a fibroblast, a precursor adipocyte, a hepatocyte, a skin keratinocyte, a mesenchymal stem cell, an adipose stem cell, various cancer cell strains, or a neural stem cell can be used.
  • PBMC peripheral blood mononuclear cell
  • an NK cell e.g., KHYG1, NK-92, YT, SNK-6, or NKL cells
  • a T cell e.g., a T cell, a precursor cell of a T cell (e.g., a hematopoietic stem cell or a lymphocyte precursor cell) or a cell population containing them
  • Any suitable T cell can be used for the first cell including but not limited a CD8-positive T cell, a CD4-positive T cell, a regulatory T cell, a cytotoxic T cell, and a tumor infiltrating lymphocyte.
  • Any suitable NK cell can be used for the first cell including but not limited to KHYG1, NK-92, YT, SNK-6, or NKL cells.
  • the first cell be collected from an animal (e.g., mammal, primate, or human), obtained by expansion culture of a cell collected from an animal (e-g ⁇ , mammal, primate, or human), established as a cell strain, or a combination thereof.
  • an animal e.g., mammal, primate, or human
  • expansion culture of a cell collected from an animal e-g ⁇ , mammal, primate, or human
  • the chimeric nucleic acid molecule can be introduced into a cell collected from the an animal (e-g ⁇ , mammal, primate, or human) itself or a conspecific an animal (e.g., mammal, primate, or human) thereof.
  • the cells in step a are NK cells (e.g., KHYG1, NK-
  • the cells in step a are T cells.
  • the first cells in step a are CD3+, and optionally stimulated with an anti-CD3 antibody, optionally in a soluble or membrane-bound form (e.g., OKT3 or mOKT3), and/or APC prior to the adding.
  • the APC are artificial APC (aAPC).
  • the aAPC express a membranous form of anti- CD3 monoclonal antibody.
  • the adding step is repeated. In other words, the adding step is repeated.
  • the adding step can be carried out 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times or until in some instances an adequate level of chimeric polypeptide expression is achieved. In other embodiments, the adding step can be carried out five times. In some embodiments, if there are multiple adding steps, they can occur on one day or they can occur on more than one consecutive day. In some embodiments, if there are multiple adding steps, they can occur on two consecutive days, three
  • the second cells e.g., a cell comprising a chimeric polypeptide, a chimeric nucleic acid molecule or both, such as, for example, a cell expressing the chimeric polypeptide
  • a predetermined antigen e.g., PD-l
  • the cells are chimeric polypeptide expressing NK cells (e.g., KHYG1, NK-92, YT, SNK-6, or NKL cells) and are optionally stimulated with irradiated cells (e.g., expressing a predetermined antigen (e-g ⁇ , PD-l)) at an effector to target ratio of 100:1, 75:1, 50:1, 25:1, 20:1, 15:1, 10:1, 9:1,
  • NK cells e.g., KHYG1, NK-92, YT, SNK-6, or NKL cells
  • irradiated cells e.g., expressing a predetermined antigen (e-g ⁇ , PD-l)) at an effector to target ratio of 100:1, 75:1, 50:1, 25:1, 20:1, 15:1, 10:1, 9:1,
  • compositions including Pharmaceutical Compositions
  • One or more chimeric polypeptides, one or more chimeric nucleic acid molecules can be part of a composition and can be in an amount (by weight of the total composition) of at least about 0.0001%, at least about 0.001%, at least about 0.10%, at least about 0.15%, at least about 0.20%, at least about 0.25%, at least about 0.50%, at least about 0.75%, at least about 1%, at least about 10%, at least about 25%, at least about 50%, at least about 75%, at least about 90%, at least about 95%, at least about 99%, at least about 99.99%, no more than about 75%, no more than about 90%, no more than about 95%, no more than about
  • a chimeric polypeptide, chimeric nucleic acid molecule, or one or more cells comprising a chimeric polypeptide, a chimeric nucleic acid molecule, or both can be part of the composition at any amount indicated herein (e.g., indicated above).
  • cells e.g., as disclosed herein
  • One or more chimeric polypeptides, one or more chimeric nucleic acid molecules can be purified or isolated in an amount (by weight of the total composition) of at least about 0.0001%, at least about 0.001%, at least about 0.10%, at least about 0.15%, at least about 0.20%, at least about 0.25%, at least about 0.50%, at least about 0.75%, at least about 1%, at least about 10%, at least about 25%, at least about 50%, at least about 75%, at least about 90%, at least about 95%, at least about 99%, at least about 99.99%, no more than about 75%, no more than about 90%, no more than about 95%, no more than about 99%, no more than about 99.
  • compositions comprising one or more chimeric polypeptides, one or more chimeric nucleic acid molecules (e.g., in the form of a bare nucleic acid molecule, a vector, a virus, a plasmid or any suitable form) or one or more cells comprising a chimeric polypeptide, a chimeric nucleic acid molecule, or both, as disclosed herein.
  • the composition is a pharmaceutical composition, such as compositions that are suitable for administration to animals (e.g., mammals, primates, monkeys, humans, canine, porcine, mice, rabbits, or rats).
  • animals e.g., mammals, primates, monkeys, humans, canine, porcine, mice, rabbits, or rats.
  • there may be inherent side effects e.g., it may harm the patient or may be toxic or harmful to some degree in some patients).
  • one or more chimeric polypeptides, one or more chimeric nucleic acid molecules can be part of a pharmaceutical composition and can be in an amount (by weight of the total composition) of at least about 0.0001%, at least about 0.001%, at least about 0.10%, at least about 0.15%, at least about 0.20%, at least about 0.25%, at least about 0.50%, at least about 0.75%, at least about 1%, at least about 10%, at least about 25%, at least about 50%, at least about 75%, at least about 90%, at least about 95%, at least about 99%, at least about 99.99%, no more than about 75%, no more than about 90%, no more than about 95%, no more than
  • a chimeric polypeptide, chimeric nucleic acid molecule e.g., added as another vector or as part of the vector comprising chimeric nucleic acid
  • one or more cells comprising a chimeric polypeptide, a chimeric nucleic acid molecule, or both can be part of the pharmaceutical composition at any amount indicated herein (e.g., indicated above).
  • cells such as cells comprising a chimeric polypeptide, a chimeric nucleic acid molecule, or both (e.g., as disclosed herein) can be part of the pharmaceutical composition at any suitable amount including but not limited to those indicated herein (e.g., indicated above).
  • the pharmaceutical composition can be presented in a dosage form which is suitable for the topical, subcutaneous, intrathecal,
  • the pharmaceutical composition can be presented in a dosage form which is suitable for parenteral administration, a mucosal administration, intravenous administration, subcutaneous administration, topical administration, intradermal administration, oral administration, sublingual administration, intranasal administration, or intramuscular administration.
  • the pharmaceutical composition can be in the form of, for example, tablets, capsules, pills, powders granulates, suspensions, emulsions, solutions, gels (including hydrogels), pastes, ointments, creams, plasters, drenches, delivery devices, suppositories, enemas, injectables, implants, sprays, aerosols or other suitable forms.
  • the pharmaceutical composition can include one or more formulary ingredients.
  • A“formulary ingredient” can be any suitable ingredient (e.g., suitable for the drug(s), for the dosage of the drug(s), for the timing of release of the drugs(s), for the disease, for the disease state, for the organ, or for the delivery route) including, but not limited to, suitable cell media, water (e.g., boiled water, distilled water, filtered water, pyrogen-free water, or water with chloroform), sugar (e.g., sucrose, glucose, mannitol, sorbitol, xylitol, or syrups made therefrom), ethanol, glycerol, glycols (e.g., propylene glycol), acetone, ethers, DMSO, surfactants (e.g., anionic surfactants, cationic surfactants, zwitterionic surfactants, or nonionic surfactants (e.g.,
  • oils e.g., animal oils, plant oils (e.g., coconut oil or arachis oil), or mineral oils), oil derivatives (e.g., ethyl oleate , glyceryl monostearate, or hydrogenated glycerides), excipients, preservatives (e.g., cysteine, methionine, antioxidants (e.g., vitamins (e.g., A, E, or C), selenium, retinyl palmitate, sodium citrate, citric acid, chloroform, or parabens, (e.g., methyl paraben or propyl paraben)), or combinations thereof.
  • oils e.g., animal oils, plant oils (e.g., coconut oil or arachis oil), or mineral oils
  • oil derivatives e.g., ethyl oleate , glyceryl monostearate, or hydrogenated glycerides
  • excipients e.g., cyste
  • the concentration of any individual formulary ingredient in a composition can be in an amount (by weight of the total composition) of at least about 0.0001%, at least about 0.001%, at least about 0.10%, at least about 0.15%, at least about 0.20%, at least about 0.25%, at least about 0.50%, at least about 0.75%, at least about 1%, at least about 10%, at least about 25%, at least about 50%, at least about 75%, at least about 90%, at least about 95%, at least about 99%, at least about 99.99%, no more than about 75%, no more than about 90%, no more than about 95%, no more than about 99%, no more than about 99.99%, from about
  • the concentration of at least one formulary ingredient is not that same as that found in the natural system in which inventive polypeptide (e.g., chimeric polypeptide) is found. In some embodiments, the concentration of at least one formulary ingredient is not that same as that found in one or more natural systems (e.g., any natural system found in nature) in which the nucleic acid molecule which encodes an inventive polypeptide (e.g., chimeric polypeptide) is found.
  • compositions can be formulated to release the active ingredient substantially immediately upon the administration or any substantially predetermined time or time after administration.
  • Such formulations can include, for example, controlled release formulations such as various controlled release compositions and coatings.
  • formulations can, in certain embodiments, include those incorporating the drug (or control release formulation) into food, food stuffs, feed, or drink.
  • Some embodiments of the invention include treatment of disease (e.g., autoimmune disease, Sjogren’s syndrome, juvenile dermatomyositis, multiple sclerosis, type-l diabetes mellitus, rheumatoid arthritis, systemic lupus erythematosus (SLE), or cancer) by administering one or more cells comprising a chimeric polypeptide, a chimeric nucleic acid molecule, or both, as disclosed herein, administering a composition as disclosed herein, or administering a pharmaceutical composition, as disclosed herein.
  • disease e.g., autoimmune disease, Sjogren’s syndrome, juvenile dermatomyositis, multiple sclerosis, type-l diabetes mellitus, rheumatoid arthritis, systemic lupus erythematosus (SLE), or cancer
  • SLE systemic lupus erythematosus
  • One or more cells comprising a chimeric polypeptide, a chimeric nucleic acid molecule, or both can be administered to animals by any number of suitable administration routes or formulations.
  • One or more cells comprising a chimeric polypeptide, a chimeric nucleic acid molecule, or both can also be used to treat animals for a variety of diseases.
  • Animals include but are not limited to mammals, primates, rodents, monkeys (e.g., macaque, rhesus macaque, or pig tail macaque), humans, canine, feline, bovine, porcine, avian (e.g., chicken), mice, rabbits, and rats.
  • the term“subject” refers to both human and animal subjects.
  • the route of administration of cells comprising a chimeric polypeptide, a chimeric nucleic acid molecule, or both can be of any suitable route.
  • Administration routes can be, but are not limited to the oral route, the parenteral route, the cutaneous route, the nasal route, the rectal route, the vaginal route, and the ocular route.
  • administration routes can be parenteral administration, a mucosal administration, intravenous administration, depot injection, subcutaneous administration, topical administration, intradermal administration, oral administration, sublingual administration, intranasal administration, or intramuscular administration.
  • the choice of administration route can depend on the cell identity (e.g., the strain, make-up, or physical and chemical properties of the cell) as well as the age and weight of the animal, the particular disease (e.g., autoimmune disease, Sjogren’s syndrome, juvenile
  • dermatomyositis dermatomyositis, multiple sclerosis, type-l diabetes mellitus, rheumatoid arthritis, systemic lupus erythematosus (SLE), or cancer), and the severity of the disease (e.g., stage or severity of disease).
  • SLE systemic lupus erythematosus
  • cancer systemic lupus erythematosus
  • combinations of administration routes can be administered, as desired.
  • Some embodiments of the invention include a method for providing a subject with a composition comprising one or more cells comprising a chimeric polypeptide, a chimeric nucleic acid molecule, or both, which comprises one or more administrations of one or more such compositions; the compositions may be the same or different if there is more than one administration.
  • diseases that can be treated in an animal include, but are not limited to autoimmune disease, allergies (e.g., IgE related allergies), asthma, Sjogren’s syndrome, juvenile dermatomyositis, multiple sclerosis, type-l diabetes mellitus, rheumatoid arthritis, systemic lupus erythematosus (SLE), cancer, blood cancer, solid tumor, leukemia, lymphoma, myeloma, breast cancer, ovarian cancer, glioblastoma, osteosarcoma, medulloblastoma, inflammatory disease, eczema, hepatitis
  • diseases that can be treated using one or more cells comprising a chimeric polypeptide, a chimeric nucleic acid molecule, or both include, but are not limited to autoimmune disease, Sjogren’s syndrome, juvenile dermatomyositis, multiple sclerosis, type-l diabetes mellitus, rheumatoid arthritis, systemic lupus erythematosus (SLE), or cancer.
  • diseases that can be treated can include autoimmune disease, allergies (e.g., IgE related allergies), asthma, Sjogren’s syndrome, juvenile dermatomyositis, multiple sclerosis, type-l diabetes mellitus, rheumatoid arthritis, or systemic lupus erythematosus (SLE).
  • diseases that can be treated can include autoimmune disease or systemic lupus erythematosus (SLE).
  • Animals that can be treated include but are not limited to mammals, rodents, primates, monkeys (e.g., macaque, rhesus macaque, pig tail macaque), humans, canine, feline, porcine, avian (e.g., chicken), bovine, mice, rabbits, and rats.
  • the term“subject” refers to both human and animal subjects. In some instances, the animal is in need of the treatment (e.g., by showing signs of disease).
  • diseases e.g., autoimmune disease, Sjogren’s syndrome, juvenile dermatomyositis, multiple sclerosis, type-l diabetes mellitus, rheumatoid arthritis, systemic lupus erythematosus (SLE), or cancer
  • an animal e.g., mammals, porcine, canine, avian (e.g., chicken), bovine, feline, primates, rodents, monkeys, rabbits, mice, rats, and humans
  • diseases e.g., autoimmune disease, Sjogren’s syndrome, juvenile dermatomyositis, multiple sclerosis, type-l diabetes mellitus, rheumatoid arthritis, systemic lupus erythematosus (SLE), or cancer
  • diseases e.g., autoimmune disease, Sjogren’s syndrome, juvenile dermatomyositis, multiple sclerosis, type-l diabetes mellitus, rheumatoid arthritis, systemic
  • one or more cells comprising a chimeric polypeptide, a chimeric nucleic acid molecule, or both can be targeted or localized to certain tissues or organs.
  • one or more cells comprising a chimeric polypeptide, a chimeric nucleic acid molecule, or both can be targeted or localized to follicular regions of secondary lymphoid tissues (e.g., by expressing of CXCR5 in the cell, by, for example, transfection or transduction).
  • treatment is to be considered in its broadest context.
  • “treating” does not necessarily imply that an animal is treated until total recovery. Accordingly, “treating” includes amelioration of the symptoms, relief from the symptoms or effects associated with a condition, decrease in severity of a condition, or preventing, preventively ameliorating symptoms, or otherwise reducing the risk of developing a particular condition.
  • reference to“treating” an animal includes but is not limited to prophylactic treatment and therapeutic treatment. Any of the compositions (e.g., pharmaceutical compositions) described herein can be used to treat an animal.
  • treating can include but is not limited to prophylactic treatment and therapeutic treatment.
  • diseases e.g., autoimmune disease, Sjogren’s syndrome, juvenile dermatomyositis, multiple sclerosis, type-l diabetes mellitus, rheumatoid arthritis, systemic lupus erythematosus (SLE), or cancer
  • treating can include but is not limited to prophylactic treatment and therapeutic treatment.
  • treatment can include, but is not limited to: preventing disease (e.g., autoimmune disease, Sjogren’s syndrome, juvenile dermatomyositis, multiple sclerosis, type-l diabetes mellitus, rheumatoid arthritis, systemic lupus erythematosus (SLE), or cancer); ameliorating or relieving symptoms of disease (e.g., autoimmune disease, Sjogren’s syndrome, juvenile dermatomyositis, multiple sclerosis, type-l diabetes mellitus, rheumatoid arthritis, systemic lupus erythematosus (SLE), or cancer); inhibiting the development or progression of disease (e.g., autoimmune disease, Sjogren’s syndrome, juvenile dermatomyositis, multiple sclerosis, type-l diabetes mellitus, rheumatoid arthritis, systemic lupus erythematosus (SLE), or cancer); reducing the severity of disease (e.g.,
  • treating does not include prophylactic treatment of disease (e.g., autoimmune disease, Sjogren’s syndrome, juvenile dermatomyositis, multiple sclerosis, type-l diabetes mellitus, rheumatoid arthritis, systemic lupus erythematosus (SLE), or cancer) (e.g., preventing or ameliorating future disease (e.g., autoimmune disease, Sjogren’s syndrome, juvenile dermatomyositis, multiple sclerosis, type-l diabetes mellitus, rheumatoid arthritis, systemic lupus erythematosus (SLE), or cancer)).
  • disease e.g., autoimmune disease, Sjogren’s syndrome, juvenile dermatomyositis, multiple sclerosis, type-l diabetes mellitus, rheumatoid arthritis, systemic lupus erythematosus (SLE), or cancer
  • Treatment of an animal can occur using any suitable administration method (such as those disclosed herein) and using any suitable amount of one or more cells comprising a chimeric polypeptide, a chimeric nucleic acid molecule, or both.
  • methods of treatment comprise treating an animal for disease (e.g., autoimmune disease, Sjogren’s syndrome, juvenile dermatomyositis, multiple sclerosis, type-l diabetes mellitus, rheumatoid arthritis, systemic lupus erythematosus (SLE), or cancer).
  • disease e.g., autoimmune disease, Sjogren’s syndrome, juvenile dermatomyositis, multiple sclerosis, type-l diabetes mellitus, rheumatoid arthritis, systemic lupus erythematosus (SLE), or cancer.
  • Some embodiments of the invention include a method for treating a subject (e.g., an animal such as a human or primate) with a composition comprising one or more cells comprising a chimeric polypeptide, a chimeric nucleic acid molecule, or both (e.g., a pharmaceutical composition) which comprises one or more administrations of one or more such compositions; the compositions may be the same or different if there is more than one administration.
  • a subject e.g., an animal such as a human or primate
  • a composition comprising one or more cells comprising a chimeric polypeptide, a chimeric nucleic acid molecule, or both (e.g., a pharmaceutical composition) which comprises one or more administrations of one or more such compositions; the compositions may be the same or different if there is more than one administration.
  • the method of treatment includes administering an effective amount of a composition comprising one or more cells comprising a chimeric polypeptide, a chimeric nucleic acid molecule, or both.
  • the term “effective amount” refers to a dosage or a series of dosages sufficient to affect treatment (e-g ⁇ , autoimmune disease, Sjogren’s syndrome, juvenile dermatomyositis, multiple sclerosis, type-l diabetes mellitus, rheumatoid arthritis, systemic lupus erythematosus (SLE), or cancer) in an animal.
  • an effective amount can encompass a therapeutically effective amount, as disclosed herein.
  • an effective amount can vary depending on the subject and the particular treatment being affected.
  • the exact amount that is required can, for example, vary from subject to subject, depending on the age and general condition of the subject, the particular adjuvant being used (if applicable), administration protocol, and the like.
  • the effective amount can, for example, vary based on the particular circumstances, and an appropriate effective amount can be determined in a particular case.
  • An effective amount can, for example, include any dosage or composition amount disclosed herein.
  • an effective amount of one or more cells comprising a chimeric polypeptide, a chimeric nucleic acid molecule, or both can be an amount of about 0.005 to about 50 mg/kg body weight, about 0.005 to about 80 mg/kg body weight, about 0.005 to about 100 mg/kg body weight, about 0.01 to about 15 mg/kg body weight, about 0.1 to about 10 mg/kg body weight, about 0.5 to about 7 mg/kg body weight, about 0.005 mg/kg, about 0.01 mg/kg, about 0.05 mg/kg, about 0.1 mg/kg, about 0.5 mg/kg, about 1 mg/kg, about 3 mg/kg, about 5 mg/kg, about 5.5 mg/kg, about 6 mg/kg, about 6.5 mg/kg, about 7 mg/kg, about 7.5 mg/kg, about 8 mg/kg, about 10 mg/kg, about 12 mg/kg, or about 15 mg/kg.
  • the dosage can be about 0.5 mg/kg human body weight, about 5 mg/kg human body weight, about 6.5 mg/kg human body weight, about 10 mg/kg human body weight, about 50 mg/kg human body weight, about 80 mg/kg human body weight, or about 100 mg/kg human body weight.
  • an effective amount of one or more cells comprising a chimeric polypeptide, a chimeric nucleic acid molecule, or both can be an amount of about 0.005 to about 50 mg/kg body weight, about 0.005 to about 100 mg/kg body weight, about 0.01 to about 15 mg/kg body weight, about 0.1 to about 10 mg/kg body weight, about 0.5 to about 7 mg/kg body weight, about 0.005 mg/kg, about 0.01 mg/kg, about 0.05 mg/kg, about 0.1 mg/kg, about 1 mg/kg, about 5 mg/kg, about 10 mg/kg, about 20 mg/kg, about 30 mg/kg, about 40 mg/kg, about 50 mg/kg, about 80 mg/kg, about 100 mg/kg, or about 150 mg/kg.
  • an effective amount of one or more cells comprising a chimeric polypeptide, a chimeric nucleic acid molecule, or both can be an amount of about 1 to about 1000 mg/kg body weight, about 5 to about 500 mg/kg body weight, about 10 to about 200 mg/kg body weight, about 25 to about 100 mg/kg body weight, about 1 mg/kg, about 2 mg/kg, about 5 mg/kg, about 10 mg/kg, about 25 mg/kg, about 50 mg/kg, about 100 mg/kg, about 150 mg/kg, about 200 mg/kg, about 300 mg/kg, about 400 mg/kg, about 500 mg/kg, about 600 mg/kg, about 700 mg/kg, about 800 mg/kg, about 900 mg/kg, or about 1000 mg/kg.
  • the dosage can be about 5 mg/kg human body weight, about 10 mg/kg human body weight, about 20 mg/kg human body weight, about 80 mg/kg human body weight, or about 100 mg/kg human body weight.
  • an effective amount of one or more cells comprising a chimeric polypeptide, a chimeric nucleic acid molecule, or both can be an amount of about 1 to about 1000 mg/kg body weight, about 5 to about 500 mg/kg body weight, about 10 to about 200 mg/kg body weight, about 25 to about 100 mg/kg body weight, about 1 mg/kg, about 2 mg/kg, about 5 mg/kg, about 10 mg/kg, about 25 mg/kg, about 50 mg/kg, about 80 mg/kg, about 100 mg/kg, about 150 mg/kg, about 200 mg/kg, about 300 mg/kg, about 400 mg/kg, about 500 mg/kg, about 600 mg/
  • “Therapeutically effective amount” means an amount effective to achieve a desired and/or beneficial effect (e.g., decreasing T FH ).
  • a therapeutically effective amount can be administered in one or more administrations.
  • a therapeutically effective amount is an amount appropriate to treat an indication (e.g., to treat disease, such as for example, autoimmune disease, Sjogren’s syndrome, juvenile dermatomyositis, multiple sclerosis, type-l diabetes mellitus, rheumatoid arthritis, systemic lupus erythematosus (SLE), or cancer).
  • treating an indication is meant achieving any desirable effect, such as one or more of palliate, ameliorate, stabilize, reverse, slow, or delay disease (e.g., autoimmune disease, Sjogren’s syndrome, juvenile dermatomyositis, multiple sclerosis, type-l diabetes mellitus, rheumatoid arthritis, systemic lupus erythematosus (SLE), or cancer) progression, increase the quality of life, or to prolong life.
  • autoimmune disease e.g., Sjogren’s syndrome, juvenile dermatomyositis, multiple sclerosis, type-l diabetes mellitus, rheumatoid arthritis, systemic lupus erythematosus (SLE), or cancer
  • SLE systemic lupus erythematosus
  • Such achievement can be measured by any suitable method, such as but not limited to measurement of the amount of TFH cells in a subject, decrease in fever, decrease of facial rash, or the extent of cancer growth
  • treatment of a disease can be a result of the activation of a cell comprising a chimeric polypeptide, a chimeric nucleic acid molecule, or both.
  • activation e.g., and certain forms or types of activation
  • activation can depend on the type of cell (e.g., T cell vs. NK cell) and the specific intracellular segment of the chimeric polypeptide.
  • activation can result in one or more of release of a cytokine (e.g., cytotoxic cytokine), degranulation, improvement of cell proliferation rate, or change in a cell surface molecule.
  • release of a cytotoxic cytokine from the activated cell can cause destruction of a target cell (e.g., TFH) expressing an antigen (e.g., PD-l).
  • a target cell e.g., TFH
  • an antigen e.g., PD-l
  • release of a cytokine or change in a cell surface molecule can stimulate other immune cells (e.g., B cell, dendritic cell, NK cell, or a macrophage), which can, in some instances, assist in the treatment of the disease.
  • other treatments of disease e.g., autoimmune disease, Sjogren’s syndrome, juvenile dermatomyositis, multiple sclerosis, type-l diabetes mellitus, rheumatoid arthritis, systemic lupus erythematosus (SLE), or cancer
  • inventive treatments described herein e.g., administering one or more one or more cells comprising a chimeric polypeptide, a chimeric nucleic acid molecule, or both, as disclosed herein,).
  • autoimmune disease e.g., autoimmune disease, Sjogren’s syndrome, juvenile dermatomyositis, multiple sclerosis, type-l diabetes mellitus, rheumatoid arthritis, systemic lupus erythematosus (SLE), or cancer
  • SLE systemic lupus erythematosus
  • cancer can include any known treatment that is suitable to treat the disease (e.g., autoimmune disease, Sjogren’s syndrome, juvenile dermatomyositis, multiple sclerosis, type-l diabetes mellitus, rheumatoid arthritis, systemic lupus erythematosus (SLE), or cancer).
  • additional optional treatments can also include one or more of surgical intervention, hormone therapies, immunotherapy, administration of NSAIDs, administration of immune- suppressing drugs, and adjuvant systematic therapies.
  • NK-92 ATCC CRL-2407 TM
  • Raji ATCC CCL-86 TM
  • S2 cells were obtained from ThermoFisher (R69007) and cultured according to their standard protocol (MARCH et al. (2010)“Use of transfected Drosophila S2 cells to study NK cell activation” Methods Mol. Biol., Vol. 612, pp.
  • streptomycin lmM sodium pyruvate, lOmM HEPES buffer, IX MEM non-essential amino acids, and O.lmM 2-beta-mercaptoehtanol (henceforth referred to as“cRPMI”).
  • Fluorescent cell images were taken using a Nikon Eclipse Ti microscope, equipped with Zyla sCMOS camera (Andor) and 488nm filter cube, and processed using NIS-Elements Imaging software (Nikon). All cell enumerations were obtained with a hemocytometer using Trypan Blue exclusion. [00114] Collection & processing of human tissue samples
  • CCHMC Children’s Hospital Medical Center
  • Tonsil tissue was processed via mincing with scissors followed by transfer of up to 4g of tissue to a gentleMACS C tube (Miltenyi Biotec) containing 8mL of phosphate-buffered saline (PBS) with 0.5mg/mL collagenase D and 3000U/mL DNasel, then dissociated on a GentleMACS Octo Dissociator (Miltenyi Biotec) using“program C”. Homogenates were incubated in a 37°C water bath for 15 minutes, then dissociated again using“program C” and transferred through a lOOum cell strainer into cRPMI. This cell suspension was then layered over Ficoll-Paque PLUS (GE Healthcare) and subjected to density-gradient centrifugation to isolate tonsillar mononuclear cells.
  • a gentleMACS C tube containing 8mL of phosphate-buffered saline (PBS) with 0.5mg/mL collagenase D and 3000U
  • PBMC Peripheral Blood Mononuclear Cells
  • PBMC peripheral blood mononuclear cells
  • PBMC and tonsil mononuclear cells were cryopreserved in FBS containing 10% dimethyl sulfoxide and frozen @ -l°C/min using CoolCell freezing containers to -80°C, then stored long-term in liquid nitrogen. Cryopreserved cells were rapidly thawed and transferred into cRPMI before use.
  • Cytofix/Cytoperm (BD Biosciences) for 20 minutes at 4°C, followed by staining in lOOpL lx Perm/Wash buffer (BD Biosciences) containing fluorescently-conjugated antibodies, each at the manufacturer’s recommended concentration, for 20 minutes at 4°C. All fixed cells were washed twice with FACS buffer to remove fixative, and kept at
  • CAR is an example of a chimeric polypeptide and the CAR’s encoding nucleic acid is an example of a chimeric nucleic acid molecule.
  • plasmid amplifications were performed by transformation and expansion of the competent E. coli strains DH5a or XLlO-Gold (for pUC57 plasmids), and Stbl3 or StellarComp (for pLVX-IRES-ZsGreenl, PiggyBac, and pAc/V5-His plasmids), grown in LB broth/agar plates containing the appropriate selective antibiotic (50pg/mL ampicillin or l00pg/mL kanamycin). Digestions were performed according to the relevant New England BioLabs online NEBcloner protocol. Digestion products were resolved using 1% agarose gel electrophoresis.
  • DNA-containing gel fragments were purified using the GeneJET Gel Extraction kit (ThermoFisher). Ligations were performed for two hours at room temperature using T4 DNA ligase (ThermoFisher). Final plasmids were purified using QIAprep Mini, Midi, or Maxi kits (Qiagen) and stored at -20°C. Plasmid insertions were verified via Sanger sequencing using custom primers (IDT Technologies or Invitrogen). Sequence files were aligned using Snapgene and/or
  • the 2 nd generation CAR (R ⁇ E1-E028-E03z) was designed by splicing the PD-L1 signal and extracellular sequences (amino acid 1-238, NP_054862, SEQ ID NO:6, and nucleic acid sequence is SEQ ID NO:33; the full amino acid sequences for PD-L1 is SEQ ID NO:38) to typical 2 nd generation CAR sequences (GETEDAN et al. (2019)“Engineering and design of chimeric antigen receptors” Mol. Ther. Methods Clin. Dev., Vol. 12, pp. 145-146, which is incorporated herein by reference in its entirety; HAN et al.
  • CD28 transmembrane & intracellular sequences CD28 transmembrane & intracellular sequences, and CD3z intracellular sequences (Figs. 1D and 1E).
  • the CAR sequence was synthesized by Genewiz into a pUC57 vector.
  • the CAR construct was excised from pUC57 and ligated into the multiple cloning site (MCS) of the lentiviral vector pLVX-IRES-ZsGreenl (Clontech) (SEQ ID NO:36).
  • MCS multiple cloning site
  • Clones were screened for the correct restriction digestion pattern and sequence- verified (see primers in SEQ ID NO:40, SEQ ID NO:4l, and SEQ ID NO:42) prior to being amplified and purified.
  • the full length, human PD-l protein sequence was obtained from UniProt (a.a. 1-288) (SEQ ID NO:34).
  • ORF open reading frame for this amino acid sequence was optimized for a Drosophila expression system using the“codon optimization tool” (IDT Technologies) (SEQ ID NO:35), then this DNA sequence was synthesized by Genewiz into a Puc57 plasmid.
  • the PD-l ORF was excised from pUC57 and ligated into the MCS of a pAc/V5-His A vector (Invitrogen), then verified via sequencing (see primers in SEQ ID NO:43 and SEQ ID NO:44), amplified, and purified (referred to as“pAc/V5-His-PDl” - SEQ ID NO:37).
  • the human PD-l ORF was excised from the aforementioned pUC57 plasmid using and ligated into the MCS of a PiggyBac Transposon, Cloning and
  • lentivirus was packaged by transfection of 293 -T cells with a 3 rd generation packaging system: pCDNA3.g/p.4xCTE plasmid (GagPol, 8pg/plate), pRSV rev plasmid (Rev, 6.5pg/plate), vector plasmid (8pg/plate), and m75-VSVG plasmid (VSV-G envelope, 2pg/plate).
  • Viral supernatant from four lO-cm plates was collected 24-48 hours post-transfection, purified via sucrose-gradient, and titer analysis was performed. Viral supernatant was concentrated to 350 pL and stored at -80°C in 25 pL aliquots. Titer was determined by transfection of control cell line and flow cytometry analysis.
  • NK-92 were transduced in 48 or 96-well flat bottom plates, previously coated overnight with human fibronectin at 20 pg/mL.
  • Cells were transduced with either CAR-containing pLVX-IRES-ZsGreen lentiviral vector or an empty-vector control lentivirus at a multiplicity of infection of 5 in 8 pg/mL protamine sulfate for 4-6 hours at 37°C. After 48 hours, cells were sorted based on fluorescent reporter expression.
  • S2 cells were chemically transfected (CaCl 2 ) using the DES Blasticidin Support Kit (Invitrogen) protocol. Briefly, the pAc/V5-His-PDl plasmid (or empty- vector control plasmid) was introduced at a ratio of >19:1 to the pCoBlast plasmid vector (Invitrogen). Cells were cultured for a week in the presence of blasticidin to select for pCoBlast-expressing clones. Blasticidin-resistant cells transfected with pAc/V5-His-PDl were stained for PD-l expression and sorted for PD-1+ cells (see Fig. 2G).
  • a portion of PD-l + S2 cells were stained for PD-l expression and further sorted into“R ⁇ -1 H ” and“PD-l Lo ” populations (see Fig. 2G). All sorted S2 cells were cultured for >2 weeks prior to co-culture with NK-92 to minimize any effects of residual aPD-l antibody still bound to S2 cell surfaces.
  • Raji cells were co-transfected via electroporation with either the empty PiggyBac plasmid (PB513) or PB513-PD1 in addition to the Super PiggyBac transposase plasmid (System Biosciences, PB210PA-1) at a 1:2.5 ratio using the Neon Transfection System (Invitrogen, MPK5000). Electroporation parameters and cell concentration were obtained from the Neon Transfection System standard protocol for Raji cells (using 100m L tips). Transfected Raji cells were cultured in 6-well, low adherent plates prior to fluorescent sorting and subsequent puromycin selection.
  • NK-92 total RNA was extracted per RNeasy Mini Kit manufacturing protocol (Qiagen). Reverse transcription was performed using iScript Reverse
  • ThermoFisher An ABI 7500 Real-Time PCR Thermal Cycler (ThermoFisher) was used under the PCR polymerase activation and amplification conditions of 95 °C for 3 minutes and 40 cycles (95 °C for l5s and 60°C for 1 minute).
  • rhPD-l-Fc, anti-PD-Ll, IgGl-Fc, and Goat IgG were solubilized in PBS and added at varying concentrations in 100m L/wcl 1 to 96-well MaxiSorp plates (ThermoFisher) before overnight incubation at 4°C. Plates were washed with PBS before counted NK-92 cells were added at a concentration of 4xl0 4 cells/mL in 200pL per well and incubated for 4 hours at 37°C. Degranulation was measured via addition of 2pg/mL of anti-CD 107 a PE-Cy7 (Biolegend) for the duration of the assay. PMA and ionomycin were added at lpg/mL each for the duration of the assay.
  • NK-92:S2 co-culture assays NK-92 and S2 cells were combined at a ratio of 1:5 (2xl0 4 and lxlO 5 , respectively) in 200pL of S2 media (without blasticidin) in 96-well round-bottom plates, then incubated at room temperature for 4 hours prior to analysis.
  • NK-92:Raji co-culture assays NK-92 and Raji cells were combined at either a ratio of 1:5 (2xl0 5 and lxlO 5 , respectively) to assess degranulation, or a ratio of 20:1 (2xl0 5 and lxlO 4 , respectively) to assess killing. Assays were carried out in in 200pL of Raji media (without puromycin) in 96-well round-bottom plates, then incubated at 37°C for 4 hours prior to analysis.
  • NK-92:CD4 co-culture assays human tonsil cells were subjected to CD4 T cell negative magnetic isolation (StemCell Technologies), achieving 88% purity, on average (data not shown), then counted. This isolated CD4 fraction was counted and cultured in 96 well round-bottom plates at 30,000 cells/well in lOOpL of cRPMI. l.5xl0 5 control or CAR NK-92 were added in lOOml cRPMI to each well for a 5: 1 ratio of NK:T cells, whereas control wells received lOOpL of cRPMI alone. Cells were then incubated for 4 hours at 37°C prior to collection and analysis.
  • NK-92:T FH :B cell co-culture assays in Figs. 4A-C human tonsil cells were subjected to either CD4 T cell or B cell negative magnetic isolations (StemCell Technologies), achieving >80% purity (data not shown).
  • the isolated CD4 T cell fraction was stained with fluorescently-conjugated antibodies to identify and sort for live
  • each well of T FH cells was combined with a single well of memory B cells (achieving a 1:2 ratio of T:B cells) and further cultured.
  • l.5xl0 5 control or CAR NK-92 were added in 50ml cRPMI to each well to achieve a 5:1 ratio of NK:T FH cells, whereas control wells received 50pL of cRPMI alone. Co-cultures were then incubated for 4 hours at 37°C prior to collection and analysis.
  • Figs. 4D-E human tonsil cells were subjected to CD4 T cell negative magnetic isolation (StemCell Technologies), achieving 88% purity, on average (data not shown), then counted.
  • This CD4 fraction was cultured in 96 well round-bottom plates at 30,000 cells/well in lOOpL of complete media.
  • l.5xl0 5 control or CAR NK-92 were added in lOOml complete media to each well for a 5:1 ratio of NK:T cells, whereas control wells received lOOpL of media alone.
  • NK-92-depleted CD4 T cells were added to CellTrace-labeled B cells at a 1:2 ratio of
  • T:B cells and cultured in complete media containing lpg/mL SEB (EMD Millipore) in 96 well round-bottom plates for 5 days. Supernatants were collected and tested for the presence of human IgM using a Human IgM ELISA kit (Thermo Fisher). At day 5, cells were collected and stained, and B cell proliferation was indicated by loss of CellTrace expression when examined via flow cytometry.
  • EMD Millipore EMD Millipore
  • Control or CAR NK-92 were added in lOOml cRPMI to wells at varying E:T ratios, whereas control wells received lOOpL of cRPMI alone. These co-cultures were carried out for 4 hours at 37°C prior to collection and analysis.
  • degranulation was assessed by addition of 2pg/mL of anti-CD 107 a PE-Cy7 (Biolegend) for the duration of the assay.
  • PMA and ionomycin were added at lpg/mL each for the duration of the assay.
  • propidium iodide (Biolegend) was added at a 1:10 ratio (volume: volume) with stained, unfixed cells approximately 10-15 minutes prior to cytometric acquisition.
  • PD-1 is a selective marker of human TFH cells
  • the PDCD1 gene encoding PD-l is expressed at low to intermediate levels on several types of leukocytes, but is expressed at higher levels by bona fide TFH cells.
  • Flow cytometer-acquired cell-surface expression of PD-l on resting, non-inflamed human tonsil mononuclear revealed that electronically gated TFH cells (CD3 + CD4 + CXCR5 + ICOS + ) exhibit the highest degree of PD-l expression relative to other major tonsillar leukocyte subsets (Fig. 1A).
  • the geometric mean fluorescence intensity (MFI) of PD-l expression on TFH cells exceeded that of regulatory T cells (Treg, CD3 + CD4 + CD25 + FoxP3 + ); naive (CD45RA + CD45RO neg ) or memory (CD45RA neg CD45RO + ) T cells (CD3 + , CD4 + or CD8 + ); immature (CD2l + CD38 hi CD27 neg IgM + IgD neg ), mature (CD27 + IgD + IgM + ), memory (CD2l + CD27 + CD38 neg ), and follicular (CD2l + CD38 10 CD27 neg IgD + IgM neg ) B cells (CD3 neg CDl9 + CD20 + ); plasmablasts (CD3 neg CDl9 + CD27 + CDl38 + CD38 hi ); NK (CD45 + CD3 neg CD56 + ) or natural killer T (NKT; CD45 + CD3 + CD56 + ) cells; classical (CDl6 neg ) and
  • PD-Ll-based CAR generation and expression on NK cells were only surpassed by the closely-related, albeit >1, 600-fold less abundant (data not shown) follicular regulatory T cell (TFR, CD3 + CD4 + CXCR5 + ICOS + CD25 + Foxp3 + ) population (Fig. 1B).
  • TFR follicular regulatory T cell
  • TFH cells demonstrated roughly 6- to 600-fold higher PD-l expression (by MFI) than most other leukocytes.
  • PD-L1 for PD-l relative to the scFv of an anti-PD-l antibody, would permit more selective targeting of PD-l hlgh TFH as compared to PD-l low cells
  • PD-l low cells are cells that express lower amounts of PD-l compared to TFH cells, such as bystander cells.
  • selective targeting of PD-l hlgh TFH cells may be achieved by optimizing the affinity of a CAR (e.g., via PD-L1 or a fragment or variant thereof) to limit its activation by PD-l low cells.
  • PMA phorbol myristate acetate
  • Fig. 2A ionomycin
  • PD-L1 CAR NK cells respond to cell-associated PD-1 via
  • NK-92 cells did not degranulate when co-cultured with S2 cells, regardless of CAR expression (Fig. 2C).
  • CAR NK-92 cells degranulated 34% more (Fig. 2D) during co-culture with sorted“PD-l 11 ”’ S2 cells (Fig.
  • Non-T FH CD4 memory T cells also exhibited increased loss of viability when cultured with CAR NK-92, though overall PI uptake in this population was lower than that of TFH cells (Fig. 3B).
  • Naive CD4 T cells demonstrated no such loss of viability over background, despite the addition of either type of NK-92 (Fig. 3B).
  • Fig. 3C shows that the CAR NK-92 cells do not kill Treg cells.
  • NK-92 effector (NK-92) to target (TFH) ratio
  • E:T target
  • CAR NK-92 demonstrated l2-fold more degranulation than control NK-92
  • Fig. 4A Calcein-AM labeled TFH and B cells co-cultured with CAR- expressing but not control NK-92 released calcein into culture supernatant in an effector cell concentration-dependent manner (Fig. 4B).
  • PI staining of target lymphocytes revealed greater loss of viability in TFH cells cultured with CAR NK-92 cells compared to control NK-92 (Fig. 4C).
  • B cells conversely, exhibited no increased PI uptake following addition of either CAR-expressing or control NK-92 (Fig. 4C).
  • mice Eight-week-old male and female human IL-3/SCF/GM-CSF transgenic mice on an immune-deficient NOD/LtSz-SCID IL-2RG-/- (NSGS mice) background, bred in-house, were conditioned via i.p. injections of 30 mg/kg busulfan (Sigma Aldrich) and kept on doxycycline chow throughout experiments. Twenty-four hours post conditioning, mice were humanized via i.v. injection of 10-15 million density-gradient purified (RBC -depleted), T cell-depleted (magnetic column; Miltenyi Biotec) cord blood leukocytes.
  • busulfan Sigma Aldrich
  • mice were given an intraperitoneal injection of 0.5 mL pristane (Sigma Aldrich).
  • pristane Sigma Aldrich
  • Fig. 5A PD-l hlgh
  • mice also have expanded populations of B cells, hyperactive T and B cells, elevated titers of autoantibodies, increased proinflammatory cytokine expression, and progressive SLE disease symptoms including lymphopenia, pulmonary serositis, and lupus nephritis.
  • a bolus of 10 7 CAR-expressing NK92 cells was introduced into these mice by intravenous injection.
  • CD4 + CD45 + T cells and CDl9 + CD45 + B cells was evaluated in the spleen of treated mice (Fig. 5C).
  • “a” or“an” means one or more than one, unless otherwise specified.
  • “another” means at least a second or more, unless otherwise specified.
  • the phrases“such as”,“for example”, and“e.g.” mean“for example, but not limited to” in that the list following the term (“such as”,“for example”, or“e.g.”) provides some examples but the list is not necessarily a fully inclusive list.
  • the word“comprising” means that the items following the word“comprising” may include additional unrecited elements or steps; that is, “comprising” does not exclude additional unrecited steps or elements.
  • the term“about,” when referring to a value or to an amount of mass, weight, time, volume, concentration or percentage is meant to encompass variations of in some embodiments ⁇ 20%, in some embodiments ⁇ 10%, in some embodiments ⁇ 5%, in some embodiments ⁇ 1%, in some embodiments ⁇ 0.5%, and in some embodiments ⁇ 0.1% from the specified amount, as such variations are appropriate to perform the disclosed method.

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Abstract

Selon certains modes de réalisation, la présente invention concerne des polypeptides chimériques. Selon d'autres modes de réalisation, la présente invention concerne des molécules d'acide nucléique chimériques codant pour les polypeptides chimériques. Selon d'autres modes de réalisation, la présente invention concerne des vecteurs comprenant la molécule d'acide nucléique chimérique. Selon d'autres modes de réalisation supplémentaires, la présente invention invention concerne des cellules comprenant une molécule d'acide nucléique chimérique, un polypeptide chimérique, ou les deux. Selon encore d'autres modes de réalisation supplémentaires, la présente invention concerne des procédés de production des cellules. Selon certains modes de réalisation, la présente invention concerne des méthodes de traitement de maladies. D'autres modes de réalisation de l'invention sont également traités dans la description.
PCT/US2019/030803 2018-05-07 2019-05-06 Polypeptides chimériques, molécules d'acide nucléique, cellules et procédés associés Ceased WO2019217253A1 (fr)

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US12398187B2 (en) 2019-03-05 2025-08-26 Nkarta, Inc. CD19-directed chimeric antigen receptors and uses thereof in immunotherapy
US12590148B2 (en) 2018-11-26 2026-03-31 Nkarta, Inc. Methods for the simultaneous expansion of multiple immune cell types, related compositions and uses of same in cancer immunotherapy

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US12398187B2 (en) 2019-03-05 2025-08-26 Nkarta, Inc. CD19-directed chimeric antigen receptors and uses thereof in immunotherapy
CN111961651A (zh) * 2020-07-02 2020-11-20 北京佳德和细胞治疗技术有限公司 基因重组PD-1抗体自分泌型人γδT细胞及其构建方法和应用
CN111961651B (zh) * 2020-07-02 2023-05-23 北京佳德和细胞治疗技术有限公司 基因重组PD-1抗体自分泌型人γδT细胞及其构建方法和应用

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